Alessandra Di Grande, PhD Student, RCSI - Antibody Genie Young Researcher Award 2018 (PhD category)
Leukaemia is a blood cancer arising from the malignant transformation of the white blood cells. The white blood cells fight infection from bacteria and viruses within the body. The incidence rate of leukaemia in Europe is 82,274 a year with a mortality rate of 5.1, leading to 53,773 deaths(1). Ireland has the second highest incidence rate, per population, of leukaemia in Europe. T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive type of acute leukaemia resulting from the transformation of T-cell lymphocytes. T-ALL is one of the most common types of leukaemia found in children, but can occur at any age (2). Although originally, the disease was associated with a very poor prognosis, over recent decades, great strides have been made in the cure treatment, with cure rates reaching over 85% in children and roughly 50% in adults (2) . Currently the conventional therapies mainly involve intensified and combined chemotherapy, associated with high toxicity and risk for the development of secondary tumors (2). Moreover drug resistance is a problem and if some cases the leukaemia cells survive and the disease comes back. Therefore, there is a need for developing new more effective treatments.
Most of the efforts in the development of new treatments for leukaemia’s come from studying molecules inside cancer cells. However, tumour progression is driven not only by cell-intrinsic alterations but also by dynamic interactions between cancer cells and the surrounding tumour microenvironment. The tumour microenvironment refers to dynamic and interactive complex of different cell types, as well as cytokines and growth factors that sustain tumour cell growth and progression. Leukaemia cancers commonly arise in the bone marrow but they spread widely through the bloodstream and infiltrate many organs. T-ALL lymphocytes accumulate in bone marrow, peripheral blood, lymph nodes, spleen and often in the central system (3). In leukaemia’s, the microenvironment in the organs such as bone marrow (BM), spleen and thymus, is composed of malignant cells mixed in a matrix of stromal cells, which include endothelial and epithelial cells, fibroblasts, dendritic cells (DCs), macrophages, and also non-malignant lymphocyte infiltration (4) (Figure1).
Each microenvironment provides extrinsic signals, such as growth factors and cytokines that could offer a survival advantage to leukemic cells. How the different components of the microenvironment play their own roles in helping tumours to grow, and their importance is still to be fully elucidated.
Bone marrow (BM) and thymus are niches of normal hematopoiesis and the soil in which T-ALL malignancies develop. So far, many studies have supported the idea that leukemic cells interact with the BM microenvironment to regulate their proliferation and survival, suggesting a pivotal role for BM niche in the survival, growth and malignant phenotypes of leukaemia cells (5, 6). As a general point, very little is known about the role of the others sites of disease, such as the spleen on the resistance and persistence of T-ALL leukaemia. Understanding the way T-ALL leukaemia behave in the surrounding microenvironment might open the door to developing new ways to tackle the disease. It is of great importance to define the signalling events occurring in all the distinct microenvironments and thus how specific signals provided from each microenvironment can contribute to the positioning, survival, and/or proliferation of T-ALL cells in order to predict chemo protective niches for the disease.
Deregulation of cell death
Deregulation of cell death signalling is involved in tumorigenesis and confers resistance to anti-cancer drugs (7). Several studies have highlighted that haematological tumours use the antiapoptotic proteins for survival under stress signals during the oncogenic processes (8, 9). Indeed, leukemic blasts often became strongly dependent on the expression of the antiapoptotic BCL-2 proteins and inhibition of the anti-apoptotic function is sufficient to kill cells. B-cell leukaemia/ lymphoma 2 (BCL-2) family proteins are a large family of both pro-apoptotic and anti-apoptotic proteins that regulate the mitochondrial cell death pathway, through protein-protein interactions (10). Both overexpression of anti-apoptotic members and down regulation of pro-apoptotic members have been associated with several human cancers, including leukaemia. Recently Dr. T. Ni Chonghaile et al. demonstrated that T-ALL leukemias develop a dependency on anti-apoptotic BCL-2 proteins that is linked to the maturation stage of the blasts. Typical T-ALLs is mostly dependent on BCL-XL, while the early T-cell progenitor ALL (ETP-ALL) subgroup is more dependent on BCL-2 (11). The anti-apoptotic BCL-2 dependence in leukaemia is of great importance due to the development of the BH3 mimetics compounds; ABT-199, ABT-263, WEHI 539. BH3 mimetics are small molecules with drug-like properties that can inhibit the function of anti-apoptotic BCL-2 proteins and induce cell death (12). In particularly, the BH3 mimetic ABT-199, is a selective BCL-2 inhibitor and it has showed the potential to be a powerful drug in the clinic, in particular in the treatment of chronic lymphocytic leukemia. This led to the approval by the Food and Drug Administration (FDA) in 2016 (13).
Each distinct microenvironment may provide extrinsic signals regulating the expression of anti-apoptotic proteins and the proapoptotic signaling to support apoptosis evasion. So far, it is not known the molecular mechanisms of crosstalk between Bcl-2 family members in the tumor microenvironment. The main aim of my PhD is examine how the leukemic blasts interact in the sites of disease, such as bone marrow, spleen and thymus microenvironment. Hallmark of my project is investigating how BCL-2 proteins are regulated in the distinct microenvironment locations presented within the disease, both in vitro and in vivo, using genetic mouse models and patient-derived-xenografts.
Altered expression of BCL-2 family proteins
Altered expression of anti-apoptotic BCL-2 proteins in different microenvironments may provide a protective niche for the cancer cells. To study the anti-apoptotic BCL-2 dependencies we use a cutting-edge technology mitochondrial BH3 profiling. In brief, BH3 profiling is a functional assay that measures the response of mitochondria to exposure of known concentrations of synthetic BH3 peptides by measuring loss of mitochondrial membrane potential or cytochrome c release. Interestingly we found that the spleenic microenvironment affects BCL-2 expression in typical T-ALL and ETP-ALL leukemias. When an ETP–ALL cell line was co-cultured with the human splenic fibroblast (HSF) cell line we found a reduction in BCL-2 dependency and a reduced sensitivity to ABT-199 (BCL-2 specific BH3 mimetic). Remarkably, we found the inverse in the typical T-ALL cell line co-cultured with the HSF; an increase in BCL-2 dependence and an increase in sensitivity to ABT-199. Currently we are trying to identify the signalling pathways which are activated resulting in this switch in BCL-2 family dependence in the different subtypes of T-ALL. In addition, we are assessing the BCL-2 family dependence of xenograft cell lines isolated from the blood and spleen of animals to determine if this switching of BCL-2 dependence occurs in vivo.
Overall a well-defined characterization of T-ALL microenvironment and cell interactions through the identification of the survival signalling occurring in each of the distinct microenvironments could lead to the discovery of novel therapeutic targets leading to rational combinations of targeted treatments for T-ALL.
2. Pui C-H, Robison LL, Look AT. Acute lymphoblastic leukaemia. The Lancet. 2008;371(9617):1030-43.
3. Van Vlierberghe P, Ferrando A. The molecular basis of T cell acute lymphoblastic leukemia. The Journal of clinical investigation. 2012;122(10):3398-406.
4. Tlsty TD, Coussens LM. Tumor stroma and regulation of cancer development. (1553-4006 (Print)).
5. Boyerinas B ZM, Yesilkanal AE, Price TT, Hyjek EM, Sipkins DA. Adhesion to osteopontin in the bone marrow niche regulates lymphoblastic leukemia cell dormancy. . Blood. 2013;121:4821–31.
6. Chiarini F, Lonetti A, Evangelisti C, Buontempo F, Orsini E, Evangelisti C, et al. Advances in understanding the acute lymphoblastic leukemia bone marrow microenvironment: From biology to therapeutic targeting. Biochimica et Biophysica Acta (BBA) – Molecular Cell Research. 2016;1863(3):449-63.
7. Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell. 2002;108(2):153-64.
8. Del Gaizo Moore V, Schlis KD, Sallan SE, Armstrong SA, Letai A. BCL-2 dependence and ABT-737 sensitivity in acute lymphoblastic leukemia. Blood. 2008;111(4):2300-9.
9. Letai A, Sorcinelli MD, Beard C, Korsmeyer SJ. Antiapoptotic BCL-2 is required for maintenance of a model leukemia. Cancer Cell. 2004;6(3):241-9.
10. Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome 18. Proceedings of the National Academy of Sciences of the United States of America. 1985;82(21):7439-43.
11. Chonghaile TN, Roderick JE, Glenfield C, Ryan J, Sallan SE, Silverman LB, et al. Maturation stage of T-cell acute lymphoblastic leukemia determines BCL-2 versus BCL-XL dependence and sensitivity to ABT-199. Cancer discovery. 2014;4(9):1074-87.
12. Ni Chonghaile T, Letai A. Mimicking the BH3 domain to kill cancer cells. Oncogene. 2008;27 Suppl 1:S149-57.
13. Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J, et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med. 2013;19(2):202-8.