Veterinary Clinical
Pathology

Research

　A video featuring one of our research projects that was selected as one of the ‘Distinctive Research Projects’ in our school (sorry, Japanese only)

We are conducting research on molecular targeted therapy for malignant tumors in dogs and cats from various perspectives. Currently, our research is primarily focused on 1) development of strategies to overcome tyrosine kinase (TK) inhibitor resistance in mast cell tumors in dogs and 2) development of molecular targeted therapy for canine squamous cell carcinoma.

1) Development of strategies to overcome tyrosine kinase (TK) inhibitor resistance in mast cell tumors in dogs
Canine mast cell tumors are malignant tumors that primarily occur in the skin. They have a high incidence rate and are very important clinically. Generally, surgery and radiation therapy are the mainstays of treatment, but, depending on the situation, chemotherapy and TK inhibitors may be used. In canine mast cell tumors, gain-of-function mutations in the KIT gene (see the figure on the right) are observed in approximately 30% of cases. When tumor cells have such a mutation, TK inhibitors targeting KIT often show antitumor effects. However, with TK inhibitor treatment, tumor cells often acquire resistance over the course of treatment, and ultimately, the tumor becomes uncontrollable in most cases.

From our research on TK inhibitor resistance in mast cell tumors, the following mechanisms have been identified:
– A secondary mutation in the KIT gene leads to the reactivation of KIT protein.
– Activation of downstream signaling pathways of KIT occurs via the activation of alternative pathway(s).
– Overexpression of KIT due to reduced ubiquitination results in enhanced proliferation signals.
– Very small numbers of TK inhibitor-resistant tumor cell clones pre-existing within the tumor tissue expand in the presence of TK inhibitors.

In this way, tumor cells modify KIT qualitatively or quantitatively using various molecular mechanisms to evade the action of TK inhibitors, ultimately acquiring resistance to TK inhibitors. To overcome this challenge, we are currently attempting to develop novel therapeutic strategies by targeting the downstream signaling regulator of KIT, SHP2. While we are still in the early stages of this research, if successful, it may provide a new approach to overcome TK inhibitor resistance in mast cell tumors.

2) Development of molecular targeted therapy for canine squamous cell carcinoma
Canine squamous cell carcinoma is a malignant tumor that occurs in various locations such as the skin, oral cavity, nasal cavity, and tonsils. The mainstay of treatment involves surgery or a combination of surgery and radiation therapy.
owever, treatment becomes challenging in situations where complete removal is difficult or when there are metastases. In response to these challenges, we are making progress in developing innovative molecular targeted therapies for squamous cell carcinoma.
Our research on canine squamous cell carcinoma has revealed the following:
– In some cases, survivin is overexpressed, and in such cancer cells, autophagy is activated, and cell death is induced by survivin inhibitors.
– In certain instances, specific kinases play a key role in proliferation, and in such cancer cells, cell death is induced by specific TK inhibitors.
– When there are abnormalities in the control molecules of the cell cycle, molecular-targeted drugs that target the cell cycle can induce cell death in such cancer cells.

There are still many uncertainties surrounding these mechanisms. Moreover, these discoveries are primarily based on in vitro or in vivo findings using mouse models. It is essential to investigate whether these mechanisms exist in actual clinical cases and, if they do, how promising they are as treatment targets and for individualized treatments. We believe that the road to clinical application is still long, but we consider diligent research as the only way to gradually advance in the development of new treatments.

3) Others
We are also conducting research on molecular targeted therapies for malignancies such as histiocytic sarcoma and plasma cell tumors.

Research

A video featuring one of our research projects that was selected as one of the ‘Distinctive Research Projects’ in our school (sorry, Japanese only)

We have consistently conducted research activities related to immunosuppressive mechanisms in cancer using mouse models and clinical samples from companion animals. Our current research focus is centered on the development of immunotherapy aimed at targeting myeloid-derived suppressor cells (MDSCs), which play a vital role in immune evasion in dogs affected by malignant tumors. Previous studies have shown a significant increase in the percentage of these cells in the peripheral blood of dogs with malignant tumors compared to healthy dogs, with an even more pronounced increase in advanced cases. This suggests that MDSCs may significantly contribute to the immunosuppressive state observed in dogs with malignant tumors. To establish a novel cancer therapy designed to control the immunosuppressive function of these MDSCs in dogs with malignant tumors, we are currently conducting a detailed functional analysis of these cells and are actively searching for drugs capable of modulating their immunosuppressive function.

Guidance policy for graduate students

I (Makoto Bonkobara) have supervised the research of ten graduate students to date. Each graduate student possesses unique qualities and characteristics, and I make an effort to tailor my guidance to align with their individual traits. For instance, with graduate students who are eager to generate ideas and perform their research independently, I provide them with the freedom to conduct experiments while periodically checking in and offering guidance as needed. On the other hand, for those who are more cautious and meticulous, I schedule frequent meetings to discuss their results and plan the next steps. Additionally, I instruct all graduate students to present their research at conferences once they have gathered a sufficient amount of experimental data. Through conference presentations, I aim to enhance their presentation skills and ability to handle question and answer sessions.
In addition to individual meetings, we hold a weekly graduate student meeting, which includes Dr. Tamura. Unlike individual meetings, this meeting is primarily conducted in English. While neither Dr. Tamura nor I consider ourselves fluent English speakers, we have experience working as researchers at the University of Texas Southwestern Medical Center, where we realized the importance of researchers communicating in English. Drawing from those experiences, we aim to familiarize graduate students with discussions in English as much as possible, hoping that they will feel comfortable and confident communicating in English and taking on challenges abroad in the future. With a similar perspective, we encourage graduate students to present their research orally at international conferences. While not all presentations are selected for oral sessions and may end up as poster presentations, we encourage them to start with oral presentations in English.

Graduate students who have been selected for Research Fellowship for Young Scientists (Japan Society for the Promotion of Science)
DC1: two graduate students
DC2: three graduate students

Career paths of graduate students after graduation
– University faculty: five graduate students (two became university faculty after serving as research fellows at the University of Texas)
– Private company: three graduate students
– Research fellows (university): one graduate student
– Animal hospital: one graduate student

Publications

First-author publications by graduate students

1. Tani H, Miyamoto R, Nagashima T, Michishita M, Tamura K, Bonkobara M. Molecular characterization of canine SHP2 mutants and anti-tumour effect of SHP2 inhibitor, SHP099, in a xenograft mouse model of canine histiocytic sarcoma. Vet Comp Oncol. 2022. 20(1):109-117. doi: 10.1111/vco.12751.
2. Tani H, Miyamoto R, Miyazaki T, Oniki S, Tamura K, Bonkobara M. A feline case of multiple myeloma treated with bortezomib. BMC Vet Res. 2022. 18(1):384. doi: 10.1186/s12917-022-03484-1.
3. Miyamoto R, Tani H, Ikeda T, Saima H, Tamura K, Bonkobara M. Commitment toward cell death by activation of autophagy with survivin inhibitor YM155 in two canine squamous cell carcinoma cell lines with high expression of survivin. Res Vet Sci. 2021. 135:412-415. doi: 10.1016/j.rvsc.2020.10.025.
4. Tani H, Miyamoto R, Noguchi S, Kurita S, Nagashima T, Michishita M, Yayoshi N, Tamura K, Bonkobara M. A canine case of malignant melanoma carrying a KIT c.1725_1733del mutation treated with toceranib: a case report and in vitro analysis. BMC Vet Res. 2021. 17(1):147. doi: 10.1186/s12917-021-02864-3.
5. Tani H, Kurita S, Miyamoto R, Ochiai K, Tamura K, Bonkobara M. Canine histiocytic sarcoma cell lines with SHP2 p.Glu76Gln or p.Glu76Ala mutations are sensitive to allosteric SHP2 inhibitor SHP099. Vet Comp Oncol. 2020. 18(2):161-168. doi: 10.1111/vco.12524.
– Selected for the journal cover –

6. Tani H, Kurita S, Miyamoto R, Sawada H, Fujiwara-Igarashi A, Michishita M, Azakami D, Hasegawa D, Tamura K, Bonkobara M. Nimustine Treatment of 11 Cases of Canine Histiocytic Sarcoma. J Am Anim Hosp Assoc. 2020. 56(3):146. doi: 10.5326/JAAHA-MS-6959.
7. Kurita S, Miyamoto R, Tani H, Kobayashi M, Sasaki T, Tamura K, Bonkobara M. Genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib in a canine mast cell tumor cell line. J Vet Pharmacol Ther. 2019. 42(6):673-681. doi: 10.1111/jvp.12816.
8. Miyamoto R, Kurita S, Tani H, Ikeda T, Ishizaka M, Saima H, Kobayashi M, Tamura K, Bonkobara. Canine squamous cell carcinoma cell lines with high expression of survivin are sensitive to survivin inhibitor YM155. M. Vet J. 2018. 240:31-36. doi: 10.1016/j.tvjl.2018.09.001.
9. Miyamoto R, Kurita S, Tani H, Kobayashi M, Sugiura S, Shigihara K, Sato Y, Tanaka Y, Tamura K, Bonkobara M. Establishment and characterization of a cell line from a feline histiocytic sarcoma. Vet Immunol Immunopathol. 2018. 201:72-76. doi: 10.1016/j.vetimm.2018.05.011.
10. Ito K, Miyamoto R, Tani H, Kurita S, Kobayashi M, Tamura K, Bonkobara M. Effect of dasatinib in a xenograft mouse model of canine histiocytic sarcoma and in vitro expression status of its potential target EPHA2. J Vet Pharmacol Ther. 2018. 41(1):e45-e48. doi: 10.1111/jvp.12449. Epub 2017 Aug 17.
11. Kobayashi M, Kuroki S, Kurita S, Miyamoto R, Tani H, Tamura K, Bonkobara M. A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells. Oncol Rep. 2017. 38(4):2543-2550. doi: 10.3892/or.2017.5865.
12. Kuroki S, Kobayashi M, Tani H, Miyamoto R, Kurita S, Tamura K, Ono K, Washizu T, Bonkobara M. Selective growth inhibition by suppression of F1Fo ATPase in canine malignant melanoma cell lines. J Vet Pharmacol Ther. 2017. 40(1):101-104. doi: 10.1111/jvp.12336.
13. Kobayashi M, Kuroki S, Tanaka Y, Moriya Y, Kozutumi Y, Uehara Y, Ono K, Tamura K, Washizu T, Bonkobara M. Molecular changes associated with the development of resistance to imatinib in an imatinib-sensitive canine neoplastic mast cell line carrying a KIT c.1523A>T mutation. Eur J Haematol. 2015. 95(6):524-31. doi: 10.1111/ejh.12526.
14. Ito K, Kobayashi M, Kuroki S, Sasaki Y, Iwata T, Mori K, Kuroki T, Ozawa Y, Tetsuka M, Nakagawa T, Hiroi T, Yamamoto H, Ono K, Washizu T, Bonkobara M. The proteasome inhibitor bortezomib inhibits the growth of canine malignant melanoma cells in vitro and in vivo. Vet J. 2013. 198(3):577-82. doi: 10.1016/j.tvjl.2013.08.003.
15. Kobayashi M, Kuroki S, Ito K, Yasuda A, Sawada H, Ono K, Washizu T, Bonkobara M. Imatinib-associated tumour response in a dog with a non-resectable gastrointestinal stromal tumour harbouring a c-kit exon 11 deletion mutation. Vet J. 2013. 198(1):271-4. doi: 10.1016/j.tvjl.2013.05.035.
16. Ito K, Kuroki S, Kobayashi M, Ono K, Washizu T, Bonkobara M. Identification of dasatinib as an in vitro potent growth inhibitor of canine histiocytic sarcoma cells. Vet J. 2013. 196(3):536-40. doi: 10.1016/j.tvjl.2012.12.016.
17. Kobayashi M, Sugisaki O, Ishii N, Yamada O, Ito K, Kuroki S, Sasaki Y, Ono K, Washizu T, Bonkobara M. Canine intestinal mast cell tumor with c-kit exon 8 mutation responsive to imatinib therapy. Vet J. 2012. 193(1):264-7. doi: 10.1016/j.tvjl.2011.10.027.

Review・Editorial

18. Bonkobara M. Recent developments in veterinary diagnostics: Current status and future potential. Vet J. 2016. 215:1-2. doi: 10.1016/j.tvjl.2016.08.010.
19. Takeuchi Y, Bonkobara M. Receptor tyrosine kinase KIT: Prognostic and therapeutic involvement in canine mast cell tumours. Vet J. 2016. 210:5-6. doi: 10.1016/j.tvjl.2015.07.012.
20. Bonkobara M. Dysregulation of tyrosine kinases and use of imatinib in small animal practice. Vet J. 2015. 205(2):180-8. doi: 10.1016/j.tvjl.2014.12.015.

Editor

21. Bonkobara M. Editor of Special Issue: Recent Developments in Veterinary Diagnostics. The Veterinary Journal. 2016. Vol 215.

Collaborative research

22. Kobayashi M, Onozawa M, Watanabe S, Nagashima T, Tamura K, Kubo Y, Ikeda A, Ochiai K, Michishita M, Bonkobara M, Kobayashi M, Hori T, Kawakami E. Establishment of a BRAF V595E-mutant canine prostate cancer cell line and the antitumor effects of MEK inhibitors against canine prostate cancer. Vet Comp Oncol. 2023. doi: 10.1111/vco.12879.
23. Maeda S, Nakazawa M, Uchida M, Yoshitake R, Nakagawa T, Nishimura R, Miyamoto R, Bonkobara M, Yonezawa T, Momoi Y. Foxp3+ Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs. Vet Pathol. 2020. 57(4):497-506. doi: 10.1177/0300985820921535.
24. Gentilini F, Turba ME, Dally C, Takanosu M, Kurita S, Bonkobara M. The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs. BMC Vet Res. 2020. 19;16(1):64. doi: 10.1186/s12917-020-02284-9.
25. Igase M, Shousu K, Fujiki N, Sakurai M, Bonkobara M, Hwang CC, Coffey M, Noguchi S, Nemoto Y, Mizuno T. Anti-tumour activity of oncolytic reovirus against canine histiocytic sarcoma cells. Vet Comp Oncol. 2019. 17(2):184-193. doi: 10.1111/vco.12468.
26. Nakano Y, Kobayashi M, Bonkobara M, Takanosu M. Identification of a secondary mutation in the KIT kinase domain correlated with imatinib-resistance in a canine mast cell tumor. Vet Immunol Immunopathol. 2017. 188:84-88. doi: 10.1016/j.vetimm.2017.05.004.
27. Kato Y, Ochiai K, Kawakami S, Nakao N, Azakami D, Bonkobara M, Michishita M, Morimatsu M, Watanabe M, Omi T. Canine REIC/Dkk-3 interacts with SGTA and restores androgen receptor signalling in androgen-independent prostate cancer cell lines. BMC Vet Res. 2017. 13(1):170. doi: 10.1186/s12917-017-1094-4.
28. Kobayashi M, Saito A, Tanaka Y, Michishita M, Kobayashi M, Irimajiri M, Kaneda T, Ochiai K, Bonkobara M, Takahashi K, Hori T, Kawakami E. MicroRNA expression profiling in canine prostate cancer. J Vet Med Sci. 2017. 79(4):719-725. doi: 10.1292/jvms.16-0279.
29. Nakahira R, Michishita M, Kato M, Okuno Y, Hatakeyama H, Yoshimura H, Azakami D, Ochiai K, Bonkobara M, Takahashi K. Oncocytic carcinoma of the salivary gland in a dog. J Vet Diagn Invest. 2017. 29(1):105-108. doi: 10.1177/1040638716673126.
30. Omi T, Nakazawa S, Udagawa C, Tada N, Ochiai K, Chong YH, Kato Y, Mitsui H, Gin A, Oda H, Azakami D, Tamura K, Sako T, Inagaki T, Sakamoto A, Tsutsui T, Bonkobara M, Tsuchida S, Ikemoto S. Molecular Characterization of the Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) Gene Associated with the Feline AB Blood Group System. PLoS One. 2016. 11(10):e0165000. doi: 10.1371/journal.pone.0165000.
31. Ochiai K, Oda H, Shono S, Kato Y, Sugihara S, Nakazawa S, Azakami D, Michishita M, Onozawa E, Bonkobara M, Sako T, Shun-Ai L, Ueki H, Watanabe M, Omi T. Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk-3). Vet Comp Oncol. 2017. 15(4):1181-1186. doi: 10.1111/vco.12254.
32. Azakami D, Nakahira R, Kato Y, Michishita M, Kobayashi M, Onozawa E, Bonkobara M, Kobayashi M, Takahashi K, Watanabe M, Ishioka K, Sako T, Ochiai K, Omi T. The canine prostate cancer cell line CHP-1 shows over-expression of the co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α. Vet Comp Oncol. 2017. 15(2):557-562. doi: 10.1111/vco.12199.
33. Kato Y, Ochiai K, Michishita M, Azakami D, Nakahira R, Morimatsu M, Ishiguro-Oonuma T, Yoshikawa Y, Kobayashi M, Bonkobara M, Kobayashi M, Takahashi K, Watanabe M, Omi T. Molecular cloning of canine co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) and investigation of its ability to suppress androgen receptor signalling in androgen-independent prostate cancer. Vet J. 2015. 206(2):143-8. doi: 10.1016/j.tvjl.2015.08.002.
34. Ochiai K, Ishiguro-Oonuma T, Yoshikawa Y, Udagawa C, Kato Y, Watanabe M, Bonkobara M, Morimatsu M, Omi T. Polymorphisms of canine BRCA2 BRC repeats affecting interaction with RAD51. Biomed Res. 2015. 36(2):155-8. doi: 10.2220/biomedres.36.155.