A new treatment modality for oral cancer: testing in felines holds promise for humans

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Colin Ong

Druggable and undruggable targets in cancer

Drug discovery in cancer has always been arduous. A major milestone in aiding our understanding and discovery of cancer drugs was the advent of molecular biology. Technical advances within this field have enabled scientists to identify, dissect, and determine three-dimensional structures of tumorigenic proteins in cells then target these proteins with drugs.  How is this possible?  Some of these protein targets such as enzymes and receptors have been found to possess hydrophobic pockets within their three-dimensional structures.  These hydrophobic pockets may serve to function as catalytic or binding sites of these proteins.  In other words, the functions of these protein targets are mediated through chemical interactions within these pockets. These functions are executed when the protein targets bind other molecules such as substrates via these hydrophobic pockets.  One can envisage that if one can interfere or disrupt these chemical interactions within the pockets of the proteins, one can suppress tumor progression. 

Over the years, chemists have successfully designed small chemicals that can “sit” or “dock” in these hydrophobic pockets and can either block or activate the function of the protein targets.  The development of these small molecules as drugs in the 20th century form the basis of the drug discovery world.  However, analysis of these druggable protein targets revealed that these targets make up only between 1% and 2% of all known targets.¹  Other protein targets that did not possess hydrophobic pockets or whose three-dimensional structures have yet to be determined were considered undruggable. However, in recent years, new technological approaches have been developed to tackle the undruggable targets.  

STAT3: An undruggable protein target in cancer

STAT3 is a protein that is highly abundant in 95% of head and neck cancers and has been identified as a potential therapeutic target.^2-3 This protein drives cancer progression and development as well as inhibits anti-cancer immunity.⁴

How does STAT3 function in the cell?  STAT3 is a transcription factor that is activated upon phosphorylation where it forms a homodimer in the cytoplasm.  Upon dimerization, the STAT3 molecules translocate to the nucleus and bind to its target DNA sequences to activate transcription that drives cellular growth. Cancer cells that overexpress or overproduce STAT3 protein are highly dependent on the biological activities of STAT3 for their survival and growth. On the contrary, deletion of STAT3 gene or inhibition of STAT3 activity did not affect the functions of normal cells and tissues; thus, STAT3 is a lucrative target for cancer treatment.  At present, development of small molecule inhibitors targeting STAT3 has been hampered by a number of technical problems. Small molecule inhibitors tested previously displayed weak binding to STAT3, limited cell permeability, and poor bioavailability.

Targeting STAST3 with a decoy oligonucleotide containing DNA sequences of STAT3 binding elements

To date, targeting STAT3 with drugs has been challenging and there are no FDA-approved STAT3 small molecule inhibitors.  Thus, researchers have undertaken different approaches to tackle this problem of targeting STAT3.   One such strategy is the synthesis of a decoy oligonucleotide containing DNA sequences of STAT3 binding elements and introducing these molecules directly into cancer cells or into the bloodstream.  In the case of the latter, the oligonucleotides will be taken up by the cancer cells.  Once in the cytoplasm of the cancer cells, these oligonucleotide molecules bind activated STAT3 homodimers, preventing them from translocating into and binding to their DNA targets in the nucleus.  In doing so, transcription in these cancer cells is suppressed and cancer progression is impeded. 

This strategy has been proven to be successful in inhibiting tumor growth in cell culture studies such as xenograft tumors of head and neck cancers in murine models.^5,6 One key advancement worth noting is the development of a modified oligonucleotide in which the ends of the oligonucleotide decoy are linked by hexaethylene glycol groups creating a cyclic oligonucleotide decoy molecule.  This chemical modification renders the oligonucleotide resistant to degradation by nucleases in the serum and confers thermal stability.⁶

Assessing safety and efficiency of the cyclic STAT3 decoy oligonucleotide

To further develop and test this cyclic STAT3 decoy oligonucleotide (CS3D) as a potential drug for head and neck cancers, Grandis and colleagues evaluated the safety and efficacy of this oligonucleotide on mouse models and domestic cats with oral cancer in a phase 1 clinical trial.⁷

(a) Preliminary tests: Examining the effects of CS3D in murine models of head and neck cancers that are immunocompetent

Given that inactivation of STAT3 gene results in enhanced anti-tumor immunity, Grandis and co-workers first sought to determine the impact of CS3D on immunocompetent mice models of head and neck cancers.⁷

They rationalized that testing on these murine models would yield useful insights prior to their testing on cats. To generate immunocompetent mice models of head and neck cancer, they used mice head and neck cancer cell lines namely MOC1, MOC22, and mEER.  All these cell lines can grow as tumors in immunocompetent mice.

First, they examined the survival responses of these cells to CS3D exposure. In two-dimensional cell culture experiments, all three cell lines displayed a decrease in survival rates when treated with the CS3D drug.  Grandis and colleagues also confirmed that the expression of STAT3 genes, VEGF, CCND1, and Bcl-X_L, were downregulated in response to drug treatment.

To better mimic head and neck tumors, the scientists isolated tumors from each of these cell lines and grafted them subcutaneously in immunocompetent mice.  These tumor tissues, which contain tumor cells, immune cells, and other cell types found in the tumor microenvironment, were grown ex vivo as tumor organoids to recapitulate three-dimensional tumor structure.  Along with the three cell lines mentioned in the previous paragraph, they also tested a fourth head and neck mice cancer model, 4MOSC1.  Tumor organoids from all the 4 mice models were exposed to CS3D for 7 days.  Grandis and colleagues documented a significant loss in cell viability and a reduction in ATP levels in all tumor organoid models tested.  

Next, Grandis and colleagues analyzed the effect of CS3D in vivo on the 3 mice cell lines (MOC1, MOC22, and mEER) grown as subcutaneous tumors in immunocompetent mice. The rodents with tumors were injected intravenously with CS3D at a dose of 5 mg/kg or 10 mg/kg for 5 times per week.  Following 3 to 5 weeks of treatment, they observed inhibition of tumor growth in all mice models when compared to mice treated with control oligonucleotide. Induction of cell death was recorded for all tumors in these mice models.  

Grandis and co-workers also addressed the possible role of immune cells in mediating anti-tumor effects following exposure to CS3D drug.  Mice with tumors were injected with anti-CD8 or anti-IgG control antibodies for 3 days.  The mice were subsequently subjected to CS3D exposure as mentioned in the previous paragraph.  Treatment with anti-CD8 antibodies ablated the anti-cancer effects mediated by CS3D drug on tumors in these mice, suggesting that CD8+ T cells (cytotoxic T cells) play a role in the ability of CS3D to suppress tumor growth.

(b)Testing in cat cell lines and phase 1 trial on cats with oral cancer   

Feline oral cancer can serve as a model of human head and neck cancer as the progression and the spontaneous arising nature of oral cancer in cats strongly mimics human head and neck cancer development.  

Grandis and colleagues initiated their investigations into feline oral cancer by examining the effect of CS3D on feline oral cancer cell lines.⁷ In vitro experiments revealed that exposure to CS3D promoted death in these cell lines.

In the phase 1 study, cats were divided into 4 cohorts. Each cohort was administered with one of the following doses of CS3D drug: 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, or 10 mg/kg. The drug was given on days 1, 3, 5, 8, 15, and 22 and the cats were examined for up to 29 days. Out of 20 cats enrolled, 7 showed partial response or stable disease.  The remaining 13 cats exhibited disease progression. No adverse side effects were noticed except for mild anemia which developed in 12 cats. The median survival time of cats that showed partial response or stable disease was much longer than the median survival time of non-responding cats (161 days versus 57 days). Loss of cell viability was observed on day 8 (when compared to day 1) in two cats which displayed either partial response or stable disease. Following 8 days post-treatment, the Bcl-X_L transcript levels were downregulated in tumors of cats with stable disease or partial response.

Analysis of immune parameters demonstrated that lower neutrophil to lymphocyte ratios were detected in responders versus non-responders on both days 1 and 8 of drug treatment.  A lower neutrophil to lymphocyte ratio in human and neck cancers is indicative of a better prognosis.⁸ Grandis and colleagues also found a decrease in the number of regulatory CD4+ T cell (regulatory helper T cell) subsets (Fox P3+ and CD25+) on the last day of the study in cats that showed partial response or stable disease versus non-responders. These cells are known to inhibit anti-tumor immune responses. 

In conclusion, their study demonstrated that CS3D, a first-in-class decoy oligonucleotide, can potentially serve as a safe and effective treatment for head and neck cancer and that it may work alongside other agents that stimulate the immune system. The next step is to await ongoing trials of STAT3 inhibitors in human head and neck cancer patients including a study of an antisense oligonucleotide targeting STAT3, danvatirsen (NCT05814666).

Abbreviations

STAT3: Signal transducer and activator of transcription 3

CS3D: Cyclic STAT3 decoy

MOC1: Mouse oral squamous cell carcinoma 1 cell line

MOC22: Mouse oral squamous cell carcinoma 2 cell line

mEER: Mouse E6/E7/hRAS oropharynx epithelial cell line

VEGF: Vascular endothelial growth factor
CCND1: Cyclin D1

BCL-XL: B-cell lymphoma-extra large

4MOSC1: 4NQO-induced murine oral squamous cells 1

ATP: adenosine triphosphate

CD8: Cluster of differentiation 8

IgG: Immunoglobulin G

CD4: Cluster of differentiation 4

FOX P3: Forkhead box P3

CD25: Cluster of differentiation 25

Header Image Caption and Source: Vitolic M. Black and White Cat Lying On Brown Bamboo Chair Inside Room. Unsplash.com. Published January 2, 2018. Accessed March 29, 2026. https://unsplash.com/photos/black-and-white-cat-lying-on-brown-bamboo-chair-inside-room-gKXKBY-C-Dk.

Edited by Chris Wang

References   

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