Introduction

            Cancer treatment has always been a challenging field for drug discovery and development. No matter what kind of treatments you are giving, chemo-, radio-, or targeted therapies, durable responses are rare and many, if not most, of the patients relapse to metastatic diseases ^1,2. This is particularly true for bladder cancer, which accounts for more than 570,000 new cases and 200,000 deaths worldwide in 2020 ³.

            Traditionally, bladder cancer patients have been treated by surgical and pharmacological treatments ⁴. However, due to the high risk of recurrence and progression, patients need to be closely monitored, which can cause a high economic burden ⁴. In addition, the overall survival of bladder cancer patients hasn’t been significantly improved in the past several decades ⁵. Therefore, the discovery of new therapeutic options is still highly desired for bladder cancer.

How did we get started?

            This project is a multi-collaborative effort from several institutions. We were inspired by our previous studies in which we established a series of patient-derived preclinical models from bile duct cancer with the collaboration from Duke University, including cell line, organoid, and mouse models (https://www.nature.com/articles/s41698-022-00320-5) ⁶. In our models, we found one very interesting genetic alteration, an FGFR fusion, which means that part of the FGFR gene is fused with a number of other genes. And this gene fusion can disrupt the normal function of FGFR, making it oncogenic.

            We then performed high-throughput screening on our models for new therapeutic regiments and excitingly, we found a two-drug combination that can synergistically kill our cancer models: pemigatinib, an FDA-approved FGFR inhibitor, and quisinostat, a pan-HDAC inhibitor. This indicates that when the two drugs are used together, they can kill cancer more effectively and efficiently.

            Since we have been longing for new treatment options for bladder cancer and FGFR fusions are also particularly common in bladder cancer ⁷, in this study, we wanted to further examine whether or not the combination of FGFR and HDAC inhibitors are still synergistic and can kill the cancer effectively for bladder cancer with FGFR genomic alterations.

Results

            In this study, we first confirmed that the combination of erdafitinib, an FDA-approved FGFR inhibitor for bladder cancer, and quisinostat is synergistic in the bladder cancer cells with FGFR3 fusions in vitro. This very encouraging result led us to further validate the efficacy of this combination in animal studies. Utilizing the xenograft models in mice, we further demonstrated that the combination of the two drugs can significantly enhance the anti-tumor effects and prolong mouse survival, compared to each individual drug. These results confirmed that the combination of erdafitinib and quisinostat is synergistic in bladder cancer with FGFR3 fusions.

            After validating the efficacy of the combination, we next started to wonder what the molecular mechanisms behind the synergy could be. Exploring the mechanism of actions of the treatments not only can help us have a deeper understanding of the treatment we are currently using, but also may reveal some new targets for the future drug discovery and development with stronger potency and less toxicity.

            We first realized that quisinostat treatment can decrease the expression level of FGFR3, and intriguingly, we also verified that the decreased FGFR3 expression can make the cancer cells more sensitive to erdafitinib treatment, which means that this might be one of possible mechanisms behind the synergy. We later also found out that quisinostat can decrease FGFR3 expression by inhibiting the translation of FGFR3.

            Since HDAC is an epigenetic regulator, we realized that FGFR3 expression might not be the only thing that is affected by quisinostat. Therefore, we kept looking for other possible mechanisms behind the synergy. Eventually, we revealed that the expression of another gene, HDGF, can also be downregulated by quisinostat, and we also confirmed that the decreased HDGF level can also make the cancer cells more sensitive to erdafitinib. More interestingly, HDGF and FGFR3 seems to function independently with each other, since disrupting one pathway doesn’t affect the other.

            Other than FGFR fusions, FGFR3 activating mutations are also particularly common in bladder cancer ⁸. Thus, we later also confirmed that erdafitinib and quisinostat are also synergistic in bladder cancer cells with FGFR3 S249C mutation, which is the most prevalent FGFR3 activating mutation in bladder cancer ⁸. More excitingly, we also demonstrated that FGFR3 and HDGF can also be downregulated by quisinostat in the bladder cancer cells with FGFR3 S249C mutation.

Impact

            FGFR alterations are present in approximately 10%  of advanced urothelial cancer ⁷ and HDACs are also frequently overexpressed in bladder cancer ⁹. However, for FGFR inhibitors, complete responses are rare with a relatively short the median duration of response ². In addition, HDAC inhibitors seems to behave poorly as monotherapies for bladder cancer ¹⁰. More importantly, the usage of HDAC inhibitors is often limited by their toxicities ¹¹. These facts further strengthen the merits of our combination, by which we can achieve strong anti-cancer effects at lower dosages and concentrations with much less and milder toxicities.

            In our study, we also observed some toxicity of quisinostat in healthy bladder cells and mouse models at higher concentrations. But importantly, combining erdafitinib with quisinostat won’t potentiate the toxicity of quisinostat. This means that by combining the two drugs together, we can use much lower doses of each drug, and therefore, relieve the toxic effects caused by the high concentration of quisinostat while maintain a stronger tumor killing effect.

            Furthermore, we also confirmed that quisinostat can achieve synergy with erdafitinib by inhibiting FGFR3 translation. This finding helps us open up a new avenue for the treatment of bladder cancer. It is possible that combing FGFR inhibitors with compounds that can inhibit FGFR3 expression or degrade FGFR3 can also achieve synergy in tumor killing and at the same time, alleviate the side effects caused by quisinostat.

            Other than FGFR3, we also found out that quisinostat can also downregulate the expression of HDGF, which might be a second mechanism independent of FGFR3. This result leads us to speculate that HDGF may also serve as a new target for future drug discovery and development to treat bladder cancer. And our results also indicate that the combination of FGFR and HDGF inhibitors might also be synergistic in bladder cancer. In addition, HDGF might as well function as a novel biomarker for bladder cancer treatment.

            Finally, we also confirmed that the combination is synergistic in bladder cancer with not only FGFR fusions, but also FGFR activating mutations. This finding further expands our combination to treat an even broader number of cancer with aberrations in FGFR.

            In conclusion, our results provide the preclinical proof of concept for the translation of the combination of erdafitinib and quisinostat from preclinical studies into clinical trials for the treatment of bladder patients with FGFR aberrations.

References

1          Szklener, K., Chmiel, P., Michalski, A. & Mańdziuk, S. New Directions and Challenges in Targeted Therapies of Advanced Bladder Cancer: The Role of FGFR Inhibitors. Cancers (Basel) 14, doi:10.3390/cancers14061416 (2022).

2          Loriot, Y. et al. Erdafitinib in Locally Advanced or Metastatic Urothelial Carcinoma. N Engl J Med 381, 338-348, doi:10.1056/NEJMoa1817323 (2019).

3          Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71, 209-249, doi:10.3322/caac.21660 (2021).

4          Sikic, D. et al. The Prognostic Value of FGFR3 Expression in Patients with T1 Non-Muscle Invasive Bladder Cancer. Cancer Manag Res 13, 6567-6578, doi:10.2147/cmar.S318893 (2021).

5          Saginala, K. et al. Epidemiology of Bladder Cancer. Med Sci (Basel) 8, doi:10.3390/medsci8010015 (2020).

6          Lidsky, M. E. et al. Leveraging patient derived models of FGFR2 fusion positive intrahepatic cholangiocarcinoma to identify synergistic therapies. NPJ Precis Oncol 6, 75, doi:10.1038/s41698-022-00320-5 (2022).

7          Mahe, M. et al. An FGFR3/MYC positive feedback loop provides new opportunities for targeted therapies in bladder cancers. EMBO Mol Med 10, doi:10.15252/emmm.201708163 (2018).

8          Gust, K. M. et al. Fibroblast growth factor receptor 3 is a rational therapeutic target in bladder cancer. Mol Cancer Ther 12, 1245-1254, doi:10.1158/1535-7163.Mct-12-1150 (2013).

9          Poyet, C. et al. Expression of histone deacetylases 1, 2 and 3 in urothelial bladder cancer. BMC Clin Pathol 14, 10, doi:10.1186/1472-6890-14-10 (2014).

10        Suraweera, A., O'Byrne, K. J. & Richard, D. J. Combination Therapy With Histone Deacetylase Inhibitors (HDACi) for the Treatment of Cancer: Achieving the Full Therapeutic Potential of HDACi. Front Oncol 8, 92, doi:10.3389/fonc.2018.00092 (2018).

11        Cengiz Seval, G. & Beksac, M. A comparative safety review of histone deacetylase inhibitors for the treatment of myeloma. Expert Opin Drug Saf 18, 563-571, doi:10.1080/14740338.2019.1615051 (2019).