A Screening of Antineoplastic Drugs for Acute Myeloid Leukemia Reveals That Fludarabine Has Weak Immunogenic Capacity and Induces T Regulatory Cells
Authors: Ocadlikova Darina , Iannarone Clara , Redavid Anna Rita , Cavo Michele , Curti Antonio 
Published: November 2020

INTRODUCTION: Recent evidence demonstrated that the treatment of acute myeloid leukemia (AML) cells with Daunorubicin (DNR) but not Cytarabine (Ara-C), results in efficient activation of anti-leukemia T cells. This process, named as immunogenic cell death (ICD), is characterized by some specific events. In the clinical setting, chemotherapy including anthracyclines and Ara-C remains a gold standard for AML treatment. However, the probability of relapse remains elevated, particularly in elderly or prognostically "high risk" patients In the last decade, Etoposide (Eto) and Fludarabine (Flu) have been added to the standard treatment for AML to potentiate its therapeutic effect, and tested in many trials. Regarding the immunogenicity of these two drugs, too few studies are reported in recent literature, and even fewer regarding AML. We therefore studied the immunogenic potential of Eto and Flu as compared to DNR and Ara-C.
METHODS: AML cell lines HL-60 and KG-1, and primary AML cells were treated with all four drugs. Calreticulin and heat shock proteins 70/90 translocation, non-histone chromatin-binding protein high mobility group box 1 and adenosine triphosphate release were evaluated. The treated cells were then pulsed into dendritic cells and used forin vitroimmunological tests, in particular for T-cell proliferation and T regulatory cells (Tregs) induction.
RESULTS: Collectively, our data indicate that, among the drugs that have been proposed to increase the efficacy of the conventional chemotherapy backbone including DNR and Ara-C, Eto has a similar and comparable capacity to DNR in inducing both early and late ICD events. On the contrary, Flu has a low if any effect, proving similar to Ara-C.
Moreover, Eto treatment was the most powerful among the tested drugs in stimulating T-cell proliferation, thus suggesting a significant capacity to activate the immune response. On the contrary, Flu had weak immunogenic potential and can be considered a non-immunogenic chemotherapy drug. Interestingly, Flu was significantly more potent in inducing suppressive T regulatory cells compared to other drugs.
CONCLUSIONS: Taken together, the present investigation expands the knowledge on the immunogenic and tolerogenic potential of the chemotherapy drugs commonly used in the therapy of AML. Among these, important differences have been observed, indicating that, particularly in an era when immunotherapy is being included in the clinical stage of AML treatment, the immunological perspective of chemotherapy should be taken into consideration in therapy decision-making.
This research was funded by AIRC (Associazione Italiana per la Ricerca sul Cancro) 2017 IG20654. Bologna AIL (Associazione Italiana contro le Leucemie)/ Bologna Section. FATRO/Foundation Corrado and Bruno Maria Zaini-Bologna.

Disclosures
Cavo: Sanofi:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Janssen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel accomodations, Speakers Bureau;GlaxoSmithKline:Honoraria, Speakers Bureau;Karyopharm:Honoraria;Novartis:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Celgene:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel accomodations, Speakers Bureau;AbbVie:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees;BMS:Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;Amgen:Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.