Targeting autophagy complements the action of ibrutinib and venetoclax in chronic lymphocytic leukemia cells

Authors: Alma Tana Jakoš Djordjevič Scientific coordinator: prof. dr. Irena Mlinarič-Raščan, Damjan Avsec Institution: University of Ljubljana, Faculty of Pharmacy, Chair of Clinical Biochemistry

INTRODUCTION: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. In the recent years, BTK inhibitor ibrutinib, PI3K inhibitor idelalisib, and Bcl-2 antagonist venetoclax revolutionized the treatment of CLL. However, these therapies present with several drawbacks, such as severe adverse events and emergence of resistance. Therefore, the demand for novel targets is high. One of such targets is autophagy, a protective and degradative cell process that plays an important but still fairly undefined role in CLL.

AIM: To elucidate the antileukemic potential of targeting autophagy in CLL.

MATERIAL AND METHODS: Autophagy reporter cell line THP-1 Difluo hLC3 and imaging flow cytometry were used to monitor autophagic flux. Autophagy was blocked using ULK1/2 inhibitor MRT68921. Cytotoxic and synergistic effects of MRT68921 and targeted therapy in primary CLL cells (the study was approved by Slovenian National Medical Ethics Committee; 0120-136/2019/4) were evaluated using PrestoBlue assay. To identify the mechanisms of synergistic effects, PI uptake and cell cycle assay were performed using flow cytometry.

RESULTS: Using autophagy reporter cells, we showed that ibrutinib and venetoclax but not idelalisib induce autophagy. Blocking the induction of autophagy with ULK1/2 inhibitor MRT68921 augmented the cytotoxicity of ibrutinib and venetoclax in CLL cell line MEC-1 and primary CLL cells. Furthermore, MRT68921 alone was able to cause cell cycle arrest at G2/M phase, but in combination with ibrutinib and venetoclax, it acted synergistically cytotoxic through a caspase-dependent mechanism, indicating that apoptosis is the mechanism of cell death. The mean EC50 values of MRT68921 in patient-derived CLL cells (n = 13) were 3.6 μM and 2.9 μM after 24 and 48 hour treatment, respectively, demonstrating that MRT68921 acts in a concentration and time-dependent manner (p = 0.0036). Moreover, compared to peripheral blood mononuclear cells MRT68921 was more cytotoxic to CLL cells (p = 0.0013), indicating it acts selectively cytotoxic towards malignant B cells.

CONCLUSION: In the present study we demonstrated that autophagy inhibition with ULK1/2 inhibitor MRT68921 acts selectively cytotoxic towards malignant B cells and augments the action of ibrutinib and venetoclax. Thus, we recognized autophagy as an important target and inhibition of autophagy as a promising new approach for the treatment of CLL

Questions & answers

Please, tell us a little bit more about yourself. My name is Alma and I finished my Master’s degree in Pharmacy at University of Ljubljana in April 2021. I am currently living in Brussels, where I am doing a one-year Masters in International Business Economics and Management. In the near future, I plan to use my knowledge in business to realise some of my scientific research projects that I care about most.

Tell us a bit more about your research and its significance. In our research, we recognized autophagy as a new therapeutic target in chronic lymphatic leukaemia (CLL) and inhibitors of autophagy as a potential new drug for the treatment of CLL. The results of our research thus represent an encouraging first step for further research into autophagy inhibitors in the treatment of CLL. Our findings are relevant to patients for whom existing therapy is inappropriate or ineffective. The prevalence of CLL is rising due to our aging population, making this research ever more important. In your opinion, what is the benefit of joining ESSP and what advice do you have for students undertaking research in the future? I think that ESSP is a great starting point for young researchers who are starting their professional career. After investing a great deal of time and energy in your research, it is incredibly rewarding to be able to share it with a wider audience and achieve international recognition. My advice to future students is to choose a research project they are really passionate about and to take advantage of opportunities to share their work and be heard.

What was the biggest challenge while carrying out the research and how did you overcome that? The biggest challenge was the all-consuming nature of the experiments. After I treated the cells with different substances, I had to wait for precise intervals of between 24 and 72 hours to take measurements. The smallest mistake or contamination meant I had to do the experiments all over again and prolong results by a matter of days.