Do STAT3 inhibitors have potential in the future for cancer therapy?

1. STAT3 activation and its role in cancer

Signal transduction activator of transcription (STAT) 3 is a member of the STAT protein family which transduces intracellular and extracellular signals to the nucleus, controlling the expression of genes responsible for multiple physiological processes. In contrast to the transient nature of physiological STAT3 signaling observed in normal cells, STAT3 is persistently activated in many malignancies, resulting in cell proliferation, survival, angiogenesis, and tumor-mediated immune evasion. Aberrant STAT3 activation in tumors can occur by canonical signaling (Figure 1), where cytoplasmic STAT3 monomers are phosphorylated by upstream signals, namely, cytokine or growth-factor activated Janus kinases (JAK), activated growth factor receptors (e.g. epidermal growth factor receptor [EGFR]), or nonreceptor tyrosine kinases (e.g. Src). Phosphorylated STAT3 (Tyr)⁷⁰⁵ dimerize through reciprocal phosphotyrosine-SH2 linkage and translocate to the nucleus, where they promote transcription of target genes, including cell-cycle regulators, proto-oncogenes, and antiapoptotic genes. Noncanonical pathways of STAT3 signaling have also been shown to play a significant role in malignant transformation, independently of STAT3 (Tyr)⁷⁰⁵ phosphorylation. STAT3 (Ser)⁷²⁷ is located at an alternative C-terminus site, where it is phosphorylated by mitogen activated pathway (MAP) kinase, c-Jun N-terminal kinase or protein kinase C (PKC) signaling, leading to enhanced STAT3 target gene transcription. In addition, STAT3 dimer stability and activity can be modified by the reversible acetylation of the STAT3 lysine685 (K685). On the other hand, negative regulators of STAT3 are thought to be perturbed in malignancy. These include suppressors of cytokine signaling (SOCS), protein tyrosine phosphatases (PTPs), and sirtuin 1-induced K685 deacetylation [1]. STAT3 SH2 domain gain-of-function mutations have been identified as oncogenic drivers of certain rare malignancies, for example, large granular lymphocytic leukemia [2]. However, these mutations are too rare to account for the high prevalence of STAT3 activation in solid tumors. Therefore, the main pathways contributing to STAT3 activation in cancers are the increased secretion of cytokines and growth factors in the tumor microenvironment, overexpression of protein tyrosine kinases, and epigenetic modulation of negative regulators of STAT3.

Figure 1. The canonical STAT3 signaling pathway. Growth factor and cytokine stimulation of cancer cells leads to activation of intracellular tyrosine kinases and receptor-associated kinases (e.g., Janus Kinase [JAK]), respectively. STATs are recruited to activated kinases and themselves become activated through tyrosine phosphorylation. Phosphorylated STAT3s dimerize through reciprocal phosphotyrosine-SH2 linkage and translocate to the nucleus, where they promote transcription of target genes.

STAT3 is regarded as an important therapeutic target in cancer for several reasons. First, it is frequently activated in a wide range of malignancies, and in-vitro STAT3 inhibition leads to growth inhibition and apoptosis in many human cancer models [3]. Second, the inhibition of STAT3 signaling leads to selective apoptosis observed in STAT3-dependent tumor cells but not normal cells. Third, multiple oncogenic pathways converge on STAT3, hence its inhibition effectively blocks several upstream tyrosine kinases simultaneously. STAT3 has been shown to be a key mediator of the oncogenic effects of two prototypal oncogene-addicted tumors; EGFR mutation-positive non-small cell lung cancer (NSCLC) and BRAF-mutant malignant melanoma [4,5]. More recently, STAT3 activation has been identified as a mechanism of resistance in oncogene-addicted phenotypes treated with their respective oncogenic pathway inhibitors. Primary pharmacological inhibition of various oncogene-driven models (EGFR, HER2, ALK, and MET) led to therapeutic resistance through upregulated STAT3 signaling, whereas disruption of the STAT3 feedback mechanism with FGFR/JAK1 inhibition restored sensitivity to their respective pathway inhibitors [6]. This has wide-ranging therapeutic implications, since resistance to primary pharmacologic inhibition is inevitable in all oncogene-addicted tumors. Yet another noteworthy role of activated STAT3 in malignancy is its contribution to tumor immune evasion through the accumulation and activation of tolerogenic dendritic and T_reg cells, as well as the upregulation of immune checkpoint proteins, for example, CTLA-4, PD-1, and PD-L1 [7]. STAT3 therefore represents an attractive cancer immunotherapy target in this modern era of immuno-oncology.

2. Existing strategies in STAT3 inhibition

The most compelling evidence for a therapeutic role of STAT3 inhibition comes from the effective targeting of JAK/STAT signaling in patients with myeloproliferative disorders, majority of which have been shown to harbor the oncogenic JAK2 V617F mutation. These gain-of-function mutations generate constitutive activation of JAK/STAT signaling, especially through STAT3 and STAT5. A phase III randomized-controlled study of the JAK1/2 inhibitor, ruxolitinib, reported prolonged survival compared to best available therapy in primary myelofibrosis [8]. This implies that there is a subset of STAT3-driven malignancies which may be exquisitely sensitive to the effects of STAT3 inhibition [2]. Despite this paradigm shift in the management of JAK/STAT-driven hematological malignancies and multiple proof-of-concept studies spanning almost two decades, STAT3-targeted agents in solid tumors have not progressed beyond the early phases of drug development. Here, we review the existing strategies of STAT3 pathway inhibition as well as the challenges encountered in the development of this class of agents.

STAT3-targeted therapies can be either synthetic or naturally occurring, and are best categorized according to their site of action (Table 1): (i) STAT3 SH2 domain or dimerization inhibitors, (ii) upstream tyrosine kinase inhibitors (e.g., JAK and Src inhibitors), (iii) STAT3-pathway oligonucleotides and inhibitors of STAT3-DNA domain binding, and (iv) peptide-mimetics of physiological negative modulators of STAT3. Although direct targeting of the STAT3 protein is the most appealing amongst the available options, there are several challenges to this. In particular, the target is a protein–protein interaction involving a large and diffuse surface area, in contrast to the easily ‘druggable’ classic binding pocket found in receptor tyrosine kinases or other enzymatic targets [14]. Furthermore, STAT proteins share a highly homologous domain structure, making the specific targeting of STAT3 all the more challenging. The first successful attempt at disrupting STAT3:STAT3 dimerization and its downstream transcription was the discovery of a phosphopeptide inhibitor (PY*LKTK) derived from the STAT3-SH2 domain-binding peptide sequence (Table 1). However, the intrinsic pharmacokinetic (PK) properties of peptides, including poor cellular permeability and lack of stability in-vivo, have curtailed their further development. Even second-generation peptidomimetics have failed to overcome these limitations [9].

Table 1. Targeting the STAT3 Pathway.

Site of Action	Class	Preclinical Data	Clinical Data	Challenges	References
SH2 domain dimerization inhibitors	Peptides	Src-transformed	Nil	Poor cellular permeability
	(XpYL)	fibroblasts		In-vivo stability	Turkson 2004 [9]
	Peptidomimetics	Breast, NSCLC,
	(ISS610)	Src-transformed fibroblasts
	Small molecule inhibitors
	STA-21 and analogues,	Breast, sarcoma, GBM	Nil	Lack of potency and	Furqan 2013 [15]
	LLL-3	GBM		specificity	Wong 2015 [10]
	S3I-201	Breast
	Stattic	Breast, HCC, GI
	OPB-31121	Breast	Advanced	Unexpected toxicities
	OPB-51602	CRC	hematologic and	Unpredictable PK
		Liver	solid tumors	properties
		Lung	(Phase I)
	Natural compounds
	Curcumin	RCC	Nil	Lack of potency and	Chai 2016 [18]
	Curcumin analogues	Breast		specificity
	(FLLL11, FLLL12, FLLL32)	Pancreas, HCC,GI
		NSCLC
Upstream tyrosine kinase inhibitors	Small molecule inhibitors
	AZD1480 (JAK1/2)	EGFR NSCLC	Phase I	Neurologic dose-limiting toxicities	Murakami 2014 [11]
	Dasatinib (Src)	NSCLC HNSCC	Phase II	Toxicities and lack of efficacy
	Natural compounds
	Butein	HCC, breast, pancreas	Nil	Lack of potency and	Chai 2016 [18]
	Capsaicin	Prostate, gastric		specificity
STAT3 pathway oligonucleotides	Antisense oligonucleotide	Lymphoma	Phase I/II
	(mRNA)	NSCLC			Hong 2015 [12]
	STAT3 decoy oligonucleotide	NSCLC, colorectal, glioma	Phase 0	Rapid degradation, not amenable to systemic administration	Sen 2012 [13]
	(DNA-binding)
	STAT3 post-transcriptional (siRNA)	Breast, brain, SCC
STAT3 DNA-binding domain	Platinum IV compounds
	IS3295	Breast	Nil	Lack of specificity	Turkson 2004 [9]
	CPA-1	Colon
	CPA-7	NSCLC

Multiple novel small molecular inhibitors targeting the STAT3-SH2 domain have been identified through virtual screening and have demonstrated physicochemical properties which indicate their potential for clinical use (Table 1). These constitute the largest class of STAT3 inhibitors presently and include STA-21, LLL-3, curcumin, and their analogs. Numerous preclinical studies have confirmed their mode of action and downstream effects on tumor cell inhibition in a variety of cell lines and animal models. However, most of these compounds have yet to be explored in clinical studies due to concerns over their relative lack of potency and selectivity [15]. OPB-51602 and OPB-31121 are the only agents in this class to have reached early phase clinical trials in both advanced solid malignancies. Although signals of efficacy were observed in tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant NSCLC and gastrointestinal malignancies, the further development of these compounds was limited by concerns over their unpredictable PK profiles and potentially severe toxicities including lactic acidosis, peripheral neuropathy and susceptibility to opportunistic infections [10]. A possible explanation for this unusual side-effect profile is the ubiquitous expression of STAT3 within the body and its diverse physiological roles, including the modulation of mitochondrial metabolism and the immune system [16]. Second-generation OPB compounds with more favorable toxicity profiles have been identified and are currently being evaluated in early phase clinical trials.

The inhibition of upstream tyrosine kinases have led to downstream abrogation of STAT3 signaling with antitumor effect in multiple preclinical models, including prostate cancer [17]. Unfortunately, early phase clinical trials of JAK1/2 inhibitors (e.g. AZD1480) and Src inhibitors (e.g. dasatinib) have revealed limited efficacy or excessive toxicities in advanced solid tumors (Table 1) [11]. Possible explanations for this include lack of specificity of the compounds tested, pathway redundancy and off-target adverse events due to pathway cross-talk.

Novel strategies of targeting transcription factors have recently emerged and appear rather promising. These include the inhibition of transcription factor gene expression using antisense oligonucleotides, inhibition of the STAT3-DNA binding domain using decoy oligonucleotides or posttranscriptional gene-silencing using small interfering RNA (Table 1) [15]. AZD9150, an antisense oligonucleotide inhibitor of STAT3, was well-tolerated and demonstrated single-agent antitumor activity against treatment-refractory lymphomas and NSCLC in a phase I clinical trial [12]. This compound has since progressed to phase II clinical evaluation. A decoy oligonucleotide targeting the STAT3 DNA-binding domain demonstrated its desired pharmacodynamic effects when injected into head and neck malignancies in a proof-of-concept study [13]. However, the rapid degradation of decoy oligonucleotides and siRNA pose a significant therapeutic challenge, rendering them unsuitable for systemic administration. Studies have also confirmed the in-vitro efficacy of other STAT3 DNA-binding domain inhibitors, including platinum (IV) compounds [15]. However, these compounds lack specificity to STAT3 and studies informing on their pharmacology as well as suitable therapeutic doses are lacking. Novel strategies involving the activation of endogenous negative regulators of STAT3 (SOCS and PTPs) are also being explored, but remain in their infancy.

3. Expert opinion

The existing evidence strongly justifies the role of STAT3 inhibition as anticancer therapy, but the relative lack of progress in the field indicates the urgent need to re-examine our strategies in the discovery of novel direct STAT3-targeted therapeutics. With few exceptions, oncogene-addiction to single pathway STAT3 inhibition is unlikely to be clinically impactful, as activating mutations are rare. The extensive cross talk and alternative signaling pathways present in STAT3-activated malignancies potentially renders single-agent STAT3 inhibition less effective generally. In addition, it has now emerged that STAT3 can be constitutively activated by both canonical and noncanonical signaling pathways, including epigenetic mechanisms. Therefore, the definition of a STAT3-activated context based on the overexpression of tyrosine-phosphorylated STAT3 in much of the existing literature may not be broadly representative. Instead, comprehensive biomarker strategies should be undertaken to define not just the STAT3-activated state, but also the mechanism of this activation in individual tumors. This will be tremendously helpful in the future development of STAT3-directed therapies and selection of these therapies for individual patients.

Based on current knowledge of STAT3 activation, direct STAT3 small molecular inhibitors remain the best candidates; upstream regulators of STAT3 lack specificity and while there is sufficient proof-of-concept for RNA inhibition, its delivery remains technically challenging. Unfortunately, the direct STAT3 targeted agents which have been evaluated to date have been somewhat disappointing due to suboptimal potency, unfavorable PK properties, and the lack of clarity on their precise mechanisms of action. Emerging advances in technology, such as the development of platforms for high-throughput screening of protein–protein interaction inhibitors and improvements in selectivity of partner proteins, are expected to boost the discovery of novel STAT3 targeted agents. Natural pharmacological STAT3 inhibitors (e.g. curcumin and butein) inhibit STAT3 through a variety of mechanisms, including STAT3 phosphorylation, dimerization, acetylation, and DNA-binding ability (Table 1). They are regarded as attractive options because of their favorable toxicity profiles, but in reality, are nonspecific and target STAT3 indirectly [18]. The definition of an ideal STAT3 inhibitor for clinical use is likely to evolve as we gain further insights into the precise mechanisms of STAT3 activation in solid tumors.

STAT3 signaling is a key physiological pathway for normal cellular function including immune regulation, and the existence of noncanonical pathways underscore challenges of drugging this target. The pleiotropic effects of targeting STAT3 will render proof-of-concept challenging, and potentiates the risk of adverse events. In particular, STAT3 has been found to be a major regulator of cellular metabolism, both in the nucleus and mitochondria [19]. For example, STAT3 phosphorylation at residue 727 is a noncanonical pathway that activates mitochondrial oxidative phosphorylation (OXPHOS) through association with GRIM-19 protein. STAT3 knockdown in in-vitro models has been shown to inhibit OXPHOS [16]. Although OXPHOS inhibition may have desirable anticancer effects in metabolically reprogrammed cancer cells, this must be carefully balanced with the potential for mitochondrial toxicities. Neuronal toxicity and lactic acidosis induced by the shift toward aerobic glycolysis have been observed in clinical trials of JAK 1/2 inhibitors and OPB-51602, respectively [20]. Moreover, infective complications of JAK inhibitor therapy have been convincingly linked to immune cell dysfunction, highlighting the role of STAT3 as an immune modulator [21]. It remains to be seen whether advances in structural analytical tools will lead to reduction in adverse effects of these novel compounds whilst preserving their efficacy.

Early clinical data of a small molecule oral STAT3 inhibitor has shown promising signals of efficacy in EGFR-mutant NSCLC after failure of EGFR TKIs [10]. The recent discovery of STAT3 as a pathway of resistance of oncogene-driven malignancies has further strengthened the scientific rationale for use of STAT3 inhibition in this context, as well as the combination of STAT3 inhibitors with other targeted therapy in a synthetic lethality approach. This therapeutic strategy is expected to result in a broad range of clinical applications for STAT3 inhibitors. A top priority is the design of robust biomarker strategies to identify molecular contexts of susceptibility and synthetic lethal approaches in order to accelerate the development of novel STAT3-targeted therapies.

Declaration of interest

A.L. Wong, J.L. Hirpara, S. Pervaiz, J. Eu, B.C. Goh have received research funding from Otsuka Pharmaceutical Co., Ltd. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Additional information

Funding

This work was supported by the National Medical Research Council of Singapore (NMRC/CSA/021/2010), NUHS Clinician Scientist Unit, and Yong Loo Lin School of Medicine, National University of Singapore.