Development of a Novel STAT3 Inhibitor for the Treatment and Chemoprevention of Lung Cancer

Abstract

The limited successful treatment options and the development of resistance to therapy continues to make lung cancer the leading cause of cancer death in men and women in the United States and accounts for greater than 15% of all deaths from cancer. Because of its high mortality rate, designing and developing novel strategies to successfully circumvent intrinsic and acquired resistance represents a major challenge in lessening the burden of lung cancer as a disease. With the overwhelming majority (approximately 85%) of lung cancer diagnoses represented by patients who are former or current smokers, the opportunity to develop chemopreventive strategies requires preclinical evaluation of candidate agents to address the lack of strategies for lung cancer prevention. Following tobacco exposure, numerous mechanistic events such as DNA adduct formation, permanent gene mutation, and transformation, the development of lung carcinogenesis during these early events is also accompanied by carcinogen-induced immune suppression. Identification of the epidermal growth factor receptor (EGFR) sensitizing mutations (namely L858R and exon 19 in-frame deletion) in NSCLC is currently used in the clinic to select patients who would respond to EGFR-directed targeted therapy but only 10% of the patient population harbor those mutations. In spite of the initial benefits observed in patients receiving EGFR tyrosine kinase inhibitors as first or second line therapy, resistance develops in more than 50% of patients as a result of a secondary T790M mutation in EGFR rendering therapy ineffective. On the other hand, NSCLC patients without those sensitizing genetic alterations can’t benefit from targeted therapy and a sub-population of those patients harbor K-RAS mutations, especially in smokers, but unfortunately, K-RAS is currently an undruggable target. Previous analysis of NSCLC patient-derived and mouse xenograft tissue reveals the presence of signal transducer and activator of transcription 3 (STAT3) as a critical signaling node, and its constitutive activation is independent of oncogene mutation status. STAT3 is a key transcriptional activator in the initiation and pathogenesis of diverse human cancers. Its role in lung cancer can be ascribed to the functional interplay between suppressing the immune system, and induction of a gene machinery in the tumor cells that translate to critical biological events such as proliferation, survival, and anti-apoptosis. Despite understanding the biology and function of STAT3 in oncogenic signaling pathways, its lack of enzymatic activity as a transcription factor has made STAT3 elusive and “undruggable” which has particularly limited STAT3 inhibitors to research purposes. Most STAT3 inhibitors either exhibit low potency, inadequate membrane penetrance or poor stability requiring higher drug concentrations to achieve therapeutic benefits, which can cause adverse effects. Efforts to develop STAT3 therapeutics for lung cancer is been pursued from different angles but none is currently FDA-approved and early results from clinical trials is not very promising. It is therefore imperative that we continue to assess candidate STAT3 inhibitors in preclinical models of lung cancer that could be viable for clinical studies. To develop a novel approach to treat and potentially prevent lung cancer development, preclinical studies were undertaken to evaluate a double-stranded oligonucleotide molecule which mimics the STAT3 response element within the promoters region of STAT3 target genes thereby acting as a “STAT3 decoy” (S3D). S3D containing hexa-ethylene glycol molecules as linkers can be ligated to form a nuclease-resistant and more stable circular STAT3 “decoy” (CS3D). To evaluate CS3D in preclinical lung cancer models, we selected NSCLC cells that are intrinsically resistant to EGFR-targeted therapy (201T), acquire resistance to EGFR-targeted therapy (H1975), and a K-RAS-induced carcinogenic model to determine its therapeutic potential and assess its chemopreventive properties. The findings associated with CS3D were compared to an inactive circularized oligonucleotide (CS3M) harboring a single nucleotide mutation rendering it unable to bind to STAT3. We observed a greater than 90% uptake of fluorescent-labelled CS3D and CS3M by NSCLC cell lines and also detectable 48 hours post-injection via systemic delivery in various organ site such as the lung, spleen, and liver. CS3D was determined to inhibit proliferation by 50%, induce apoptosis, decrease independent-anchorage growth by 70%, and suppress c-Myc and Bcl-XL gene expression. Surprisingly, CS3D increased p-STAT3 ubiquitination thereby enhancing its degradation. This effect is accompanied by a decrease in nuclear and cytoplasmic p-STAT3 pools, suggesting a CS3D-induced degradation process. To support the preliminary in vitro findings, CS3D demonstrated a robust antitumor effect measured as a 96.5% reduction in xenograft models inherently resistant (201T) to EGFR targeted therapy and 81.7% in drug-induced resistant models (H1975). CS3D promoted an anti-proliferative phenotype measured as a decrease in Ki67 index, increase in apoptosis, and increase in lymphocyte infiltration suggesting an enhanced immune function, with no systemic toxicity observed through the course of the studies. Assessment of CS3D in a carcinogen-induced lung cancer model that induces K-RAS mutations, showed that CS3D causes a decrease in preneoplasia, tumor size, and number of tumors by 30%, 50%, and 40% respectively, highlighting the chemopreventive properties of CS3D. Early histological analysis revealed a lung microenvironment that is less primed for tumor initiation or progression because markers of angiogenesis, and cell cycle progression such as VEGF, and MYC respectively, were downregulated. In addition to key factors in survival (VEGF and MYC), markers of chronic inflammation such as NF-kB were also suppressed at 8 weeks post CS3D administration except for COX-2 which was elevated. These effects were accompanied by an increase in macrophage infiltration. However, at a later timepoint, 20 weeks post-treatment, we were able to detect higher levels of IL-6 which might point to a rescuing signaling feedback loop that could possibly lead to development of resistance to CS3D. Flow cytometry analysis to define the immune cell profile revealed that CS3D favored an antitumor immune response over an immunosuppressive TME by increasing the number of lung M1 macrophages while decreasing M2 macrophages and MDSCs. Ratio of M1 to M2 was 3:1 with CS3D and 1:3 with the control construct (CS3M). Despite the robust anti-tumor effect observed in the xenograft and chemopreventive model, CS3D also activates feedback loops by increasing IL-6, HER and FGFR receptor ligand secretion. Production of those ligands in turn activates corresponding receptors that leads to the phosphorylation of MAPK in an autocrine fashion. In combination with EGFR and FGFR inhibitors, CS3D produces anti-proliferative effects that are synergistic as a measure of proliferation, migration, and anchorage-independent growth. Confocal analysis indicates that the “STAT3 decoy” (CS3D) can successfully be incorporated into NSCLC cells and exhibit stability via systemic delivery, conferring a strong translational potential. CS3D effectively alters STAT3 function by suppressing its transcriptional activity and causing its subsequent degradation, demonstrating the ability of CS3D to act as a molecular sink for STAT3 dimers. This provides a unique feature compared to other STAT3 inhibitors which are designed to target the STAT3 phosphorylation step. The ability of unphosphorylated dimers to be transcriptional active renders phospho-STAT3 inhibitors ineffective compared to decoy oligonucleotides like CS3D. Comparison of CS3D to an oligonucleotide that differs by a single base-pair (CS3M), which showed no therapeutic effects, was imperative to confirm CS3D selectivity against STAT3.