Identification and development of small molecule therapies for the treatment of acute myeloid leukaemia

Abstract

Acute myeloid leukaemia (AML) is a cancer of the bone marrow in which immature myeloid cells exhibit uncontrolled proliferation and failure to differentiate. The global 5-year overall survival rate for AML ranges between 25 – 35% indicating the urgent need for novel therapeutic alternatives. Current therapies include bone marrow transplant (for a minority of suitable patients), chemotherapy and a handful of small molecule drugs approved for selected patient subgroups. Poor survival rates and limited therapy response are partially attributed to inter- and intra- patient heterogeneity. The concept of personalised medicine has emerged as one potential strategy to overcome the diversity of AML patients. For this approach to succeed novel small molecules with unique anti-leukemic properties must be developed to expand the arsenal of clinical alternatives. The primary objective of the thesis was to identify and develop small molecule therapies for the treatment of AML. In paper I we supported the development of novel small molecules and investigated their cytotoxic properties in AML cell lines. N-heterocyclic carbenes were generated and complexed with silver using a novel multi-step synthetic pathway to produce the putative metallodrugs, NHC-1 and NHC-2. Dose response curves were generated for each of the compounds to determine the IC50 of each compound in the AML cell lines, MOLM-13 and HL-60. Cell death induction was characterised as rapid and associated with apoptotic nuclear morphology. Interestingly, we also observed increased phosphatidylserine expression in HL-60 cells treated with the silver NHC complexes as compared with cytarabine. In paper II we identify the tryptamine derivative, Serdemetan (SDM), as a candidate for small molecule therapy in AML. SDM was originally discovered as part of an internal drug development program at Janssen Pharmaceutical, attempting to identify novel Hdm2 inhibitors for use in solid cancers. In AML cell lines and patient samples SDM activity varied independent of p53 status, suggesting the agent may have additional molecular targets. SDM was well tolerated and significantly prolonged survival in preclinical models of AML including the syngeneic BNML rat model and MOLM-13 xenograft, further indicating the agent’s suitability for development in AML. Interestingly, our studies revealed novel mechanisms of action including upregulation of autophagy markers and depletion of Akt1 expression. In paper III we propose repositioning Hydroxyurea (HU) and Valproic Acid (VPA) as a combination therapy for the treatment of AML. Both agents have previously been trailed clinically in AML and are generally well tolerated. HU and VPA combined to induce synergistic cell death in multiple AML cell lines. Unlike SDM, combination induced apoptosis and proliferation arrest appeared partially dependent on wildtype p53 expression. Mechanistic studies revealed that VPA dramatically enhances HU induced DNA double strand breaks, likely by downregulating the homologous recombination protein, Rad51. The synergistic efficacy of the combination appeared to be maintained in vivo as combination therapy was superior to monotherapies in OCI- AML3 and patient derived xenograft models of AML. Through different selection strategies the work performed in this thesis effectively identified a diverse group of small molecules worthy of further investigation for the treatment of AML. Mechanistic studies provided novel insights into how each of the agents exert their anti-leukemic properties and should guide biomarker development and identification of sensitive patient subgroups. The preclinical animal studies performed in papers II and III provide an important step towards clinical translation.