The inherited bone marrow failure syndrome Fanconi Anaemia (FA) is caused by mutations in any one of the multiple FANC genes, which encode proteins that collaborate in the FA/BRCA DNA damage response pathway. FA is characterised by extreme predisposition to acute myeloid leukaemia (AML). AML in FA is associated with typical chromosomal aberrations involving gains of the long arm of chromosome 3 (3q gains). These are linked to overexpression of the oncogene ecotropic viral integration site 1 (EVI1). Based on this clinical observation, the hypothesis that EVI1 confers leukaemic transformation, in particular in the context of FA, was tested. Mouse embryonic stem cells with either functional or disrupted FA/BRCA-pathway were used to model normal and FA-associated embryonic haematopoiesis, and the effect of EVI1 overexpression was assessed in this model. EVI1 functions were also investigated with respect to protein interactions, focusing on the interaction with the co-repressor C-terminal binding protein 1 (CtBP1), in the context of genotoxic stress. To study this, in vitro haematopoietic differentiation assays, flow cytometry, mass spectrometry, immunoprecipitations and immunofluorescence were employed. In vitro haematopoietic differentiation using mouse embryonic stem cells with defective Fanca was successfully developed and applied. The analysis revealed that EVI1 overexpression in haemangioblast-like cells prevented the generation of haematopoietic precursors through endothelial to haematopoietic transition. Studies into EVI1 protein interaction dynamics showed that DNA damage-induced phosphorylation of EVI1 modifies interaction with the co-repressor CtBP1. This interaction was demonstrated to be partially required for the EVI1-induced block of the development of haematopoietic precursors using the mESC-based model. An EVI1-mediated modulation of the FA phenotype characteristic G2-arrest and of the FA-associated embryonic haematopoiesis was not demonstrated. This study contributes to the understanding of the function of the EVI1 oncogene in normal and FA-associated haematopoiesis and the DNA damage response. FA-associated haematopoiesis and leukaemogenesis can be further studied using the embryonic haematopoiesis model developed here, and further studies can build on the data generated with respect to EVI1.

In Western Europe and North America, acute myeloid leukaemia (AML) is predominantly a disease of the elderly, with a median age at the time of presentation in excess of 60 years. However, many clinical trials in AML fail to recruit elderly adults due to a combination of strict entry criteria, or physician or patient bias. Thus, clinical outcome data from many trials may not be readily applicable to older patients with the disease. Furthermore, because the clinical outcome for many older patients with AML is frequently poor, elderly patients who receive intensive chemotherapy with curative intent are frequently selected for treatment on clinical criteria rather than on objective prognostic criteria that may define clinical outcome. The karyotype at the time of presentation may be considered one of the most important prognostic factors in adult AML. Therefore, the aim of this thesis were firstly to analyse the clinical outcome data from a cohort of elderly patients managed at a single centre in order to document the cytogenetic features of AML in an elderly population, to define the prognostic importance of presentation karyotype in the elderly, and to identify other prognostic factors. Retrospective analysis clearly demonstrated improved clinical outcome for older patients with AML over time, primarily as a consequence of improved supportive care and the delivery of more intensive chemotherapy. In addition, 'unfavourable' presentation karyotype, increasing age and raised serum LDH were found to correlate with poor clinical outcome Molecular cytogenetic techniques based upon fluorescence in-situ hybridisation technology offer the chance to detect and analyse cytogenetic aberrations at a higher resolution than can be achieved with conventional techniques. The cytogenetic data provided by comparative genomic hybridisation and mulitplex fluorescence in-situ hybridisation when used in the analysis of elderly patients with AML were found to correlate well with results obtained by conventional methods. Importantly, additive cytogenetic data were more likely to be provided if multiplex-fluorescence in-situ hybridisation was used in the analysis of cases with marker chromosomes or in cases with complex karyotype, although the technique was limited by an inability to reliably detect telomeric translocations. In addition, although both techniques can be used to complement conventional G-banding analysis, conventional FISH methods are often required to confirm the results.

The tumour microenvironment consists of an immunosuppressive niche created by the complex interactions between cancer cells and surrounding stromal cells. A critical component of this environment are myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immature myeloid cells arrested at different stages of differentiation and expanded in response to a variety of tumour factors. MDSCs exert diverse effects in modulating the interactions between immune effector cells and malignant cells. An increased presence of MDSCs is associated with tumour progression, poorer outcomes, and decreased effectiveness of immunotherapeutic strategies. In this project, we sought to quantify and characterise MDSC populations in patients with Acute Myeloid Leukaemia (AML) and delineate the mechanisms underlying their expansion. We have demonstrated that immune suppressive MDSCs are expanded in the peripheral blood and bone marrow of patients with AML. Furthermore, AML cells secrete extra-cellular vesicles (EVs) that skew the tumour microenvironment from antigen-presentation to a tumour tolerogenic environment, through the expansion of MDSCs. We then demonstrated that MDSC expansion is dependent on tumour and EV expression of the oncoproteins MUC1 and c-Myc. Furthermore, we determined that MUC1 signalling promotes c-MYC expression in a microRNA (miRNA) dependent mechanism. This observation lead us to elucidate the critical role of MUC1 in suppressing microRNA-genesis in AML, via the down-regulation of the DICER protein, a key component of miRNA processing machinery. Finally, exploiting this critical pathway, we showed that MDSCs can be targeted by MUC1 inhibition or by the use of a novel hypomethylating agent SGI-110.

Abstract In Oulu University Hospital Haematological Ward during the years 1990–1991, a manual blood culture system was able to detect bloodstream infection (BSI) in 23% of febrile episodes of patients with acute myeloid leukaemia (AML), whereas during the years 1992–1993 an automated continuous-monitoring blood culture system (CMBCS) BacT/Alert® detected BSI in 40% of febrile episodes (p = 0.043). During the years 1997–2003, regimens containing high-dose cytarabine predisposed patients to laboratory-confirmed BSI (LCBI) with an odds ratio (OR) of 2.3 (with 95% confidence interval (CI) from 1.2 to 4.2). The LCBI risk was lowest after thioguanine-containing regimens (OR 0.26, 95% CI; 0.12–0.58). In the register data (years 1992–2006) from the prospective multi-centre AML -92 trial, when compared to cycle I, the OR for LCBI was significantly higher (from 4.8 to 5.8) in subsequent cycles (p < 0.001). In all, 67% of mortality due to BSI occurred in patients with active leukaemia. An inoculum of microorganisms to produce 10 colony-forming units (cfu)/ml of 10 gram-positive coccal strains, 10 gram-negative bacillar strains and 8 Candida yeast strains was cultured in BacT/Alert® blood culture bottles in the presence of several chemotherapeutic drugs. Of the chemotherapeutic drugs tested, the anthracyclines exhibited inhibitory effects on the growth of microorganisms in concentrations corresponding to the therapeutic levels. In the standard bottles, doxorubicin increased the incubation time of gram-positive cocci and idarubicin increased the incubation time of Candida glabrata. However, no increase in the incubation time of any microbes was detected in the antimicrobial-neutralizing FAN bottles. In conclusion, the use of CMBCSs has resulted in an increased LCBI rate in neutropenic AML patients. In general, chemotherapeutic agents have no significant inhibitory effects on the growth of common microbial pathogens in blood culture. The detection of some difficult-to-culture microbial strains – C. glabrata for example – in blood cultures may be impaired by the presence of chemotherapeutics in blood. The chemotherapeutics may also affect the LCBI rate in other ways. As a predictor of adverse outcome of infection, the presence of active leukaemia is more important than the type of chemotherapy being administered.

Faculty of Science School of Molecular Medicine and Haematology Masters Dissertation / Studies on the development of drug resistance in several cancer types, including acute myeloid leukaemia (AML), have implicated the PI3-kinase pathway. This pathway phosphorylates Akt resulting in the activation of proteins involved in cell survival. The aim of this study is to determine the role that Akt plays in urvival and the relationship between Akt, IKK and IkB in HL-60 cells. This study demonstrated that etoposide caused apoptosis in HL-60 cells, which was slightly increased when the PI3-kinase pathway was inhibited by LY294002. Stimulation with PDGF resulted in cell proliferation and increased Akt, IKK and IkB phosphorylation. Although pre-treatment with LY294002 decreased the amount of Phospho-Akt, phosphorylation of IKK and IkB still occurred. Therefore additional pathways must be involved in IkB regulation in HL-60 cells. Akt mRNA transcription was decreased when the cells were pretreated with LY294002 and either PDGF or etoposide. In conclusion, the PI3-kinase pathway plays a minor role in the survival of HL-60 cells and Akt substrates other than IKK are mediating this survival.

Tyrosine kinase inhibitors (TKIs) are currently the first therapy option for chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients. However, many patients affected by CML and AML may develop resistance to TKIs or may not recover under this treatment regime. New potential and more effective treatments are recently emerging. Heat shock protein inhibitors (HSPIs) and the proteasome inhibitor Bortezomib are drugs which have been yet to be successfully tested on leukemic patients, despite being successful on other malignancies such as multiple myeloma (MM). The combination between HSPIs and Bortezomib could potentially be successful in killing leukemic cells, by enhancing their respective molecular mechanisms. Indeed, HSPIs would bind to HSP72 avoiding the protein to exert its ligase function to the proteasome, whilst Bortezomib could stop the ubiquitinated proteins to enter the proteasome and ultimately inducing apoptosis. To test the effects of such combination, cell viability was measured via MTS assay, apoptosis levels were tested through Annexin V\PI assays. Involvement of HSP72 and pro-survival protein Bcl-2 were measured via flow-cytometry. The cells were administered with HSPIs and Bortezomib first as single agents for 24 hours, to establish working minimal concentration. Also, the drugs were tested for a shorter time, to understand when the drugs start to be effective. It emerged that one hour is sufficient for the drugs to give an initial effect in terms of cell viability and apoptosis. Following, combination experiments of HSPIs and Bortezomib were performed; the first drug was administered for one hour, the second following one hour and the cells were incubated for 24 hours. This was repeated alternatively for both type of drugs on the different cell lines. MTS and Annexin V\PI showed that there is not a synergistic effect between drugs, but instead there is antagonism. No necrosis was found at any level of the study. The cells were then probed for HSP72 and Bcl-2, to investigate their involvement in apoptosis mechanisms. Following 6 hours of combined and single agent treatment, both type of drugs inhibit HSP72 but failed to reduce the expression of Bcl-2, particularly on AML cells. It is thus proposed that CML and AML cells may die by apoptosis following a short time of treatment with HSPIs and Bortezomib by an extrinsic pathway of apoptosis, independent from Bcl-2 involvement and from mitochondrial pathway of apoptosis. This study may be the first to indicate a potential use of HSPIs and Bortezomib on CML and AML patients for a short time of treatment, although not in combination. Future studies are needed to further investigate the mechanisms of action of these drugs, aiming to potentially give CML and AML patients another successful therapy option to overcome resistance to canonic chemotherapy.

The tumour microenvironment plays a key role in tumour progression. In this thesis acute myeloid leukaemia (AML) was used as a model system to investigate the interplay between stromal and cancer cells. AML is a heterogeneous clonal disorder of haematopoietic undifferentiated progenitor cells or 'blast cells', which accumulate in the bone marrow and lead to the reduced output of crucial haematopoietic elements. Due to its heterogeneity (at least in part), treatment of the disease has not witnessed great innovation in the past 30 years. The bone marrow microenvironment (BMM) has a key role in the haematological malignancies contributing to the survival of leukaemic blasts. Relapse in AML occurs because of residual disease and evidence suggests that this resistance is facilitated through leukaemic cells ability to reside in BMM niches. To understand the precise role of the BMM in AML progression and therefore target any supportive mechanisms requires knowledge of how AML cells communicate with their microenvironment. In the work presented in this thesis I undertook a multi-proteomic approach that utilised liquid chromatography tandem mass spectrometry (LC-MS/MS) to assess the interplay between AML and BMM cell signalling. This thesis shows the results of a secretomic analysis of stromal cell lines, which identified a previously uncharacterized panel of six stromal secreted proteins (BMP-1, CSF-1, CTGF, HGF, S100-A4 and S100-A11) that support primary AML cell survival and proliferation in culture. Comparison of AML cell signalling (using global phosphoproteomic methods) following treatment with the newly identified growth factors revealed that these signalling proteins elicit multi-nodular activation of signalling networks with known anti-apoptotic activity. Consistent with the cell signalling proteomics data, cell viability studies as a function of pharmacological kinase inhibitor treatment determined that the sensitivity of AML to targeted kinase inhibitors was modulated by the supportive stromal conditioned media. To investigate heterotypic signalling between cell populations, AML/stromal cell co-cultures were designed, tested and optimised. These studies identified additional activated pathways in AML cells that were only present when AML cells had physical interaction with stroma. Complementary analysis of the stromal cells which had been first cultured with AML cells revealed that despite heterogeneity there is an emerging stromal phospho-proteomic signature that is different in BMM independent AML cells vs BMM interactive AML cells. Collectively these findings evidence the influence that the BMM can have on AML signalling. Although evidence for the influence of BMM in modulating AML resistance to standard chemotherapy exists, this study highlights specific BMM components that contribute to the ability of AML cells to circumvent current treatments based on kinase targeted drugs. These observations have implications for designing future therapies for AML.

Despite high remission rate in acute myeloid leukaemia (AML) after chemotherapy, relapse of the underlying disease remains a major challenge and one of the most frequent causes of treatment failure. In this study, the presence of leukaemiaassociated phenotypes (LAPs) was first studied retrospectively using our standard diagnostic protocol with 3-colour flow cytometry. LAPs were present in 54 (64%) of 84 AML patients analysed between 2002 to 2004. The presence of LAPs was correlated with failure to respond to induction chemotherapy (p <0.05) in univariate analysis. Presence of LAPs was shown to be an independent predictor for failure to respond to induction chemotherapy with a relative risk ratio of 1.6 (p < 0.05, 95% CI, 1.0-2.6) in multivariate analysis. Subsequently, in a prospective study, we used 5-colour multiparametric flow cytometry (MFC) for detection of LAPs to determine if LAPs could be detected in a greater proportion of leukaemic patients and minimal residual disease (MRD) detection could therefore be applied in more patients. In 54 consecutive, newly diagnosed AML patients from 2005 to 2007, LAPs were identified in 51 (94%). Thus, MRD studies were potentially applicable to virtually all patients. The sensitivity and specificity of MFC technique was improved by analysing 10 normal and 5 regenerating bone marrows (BM) for the presence of these LAPs and by determining maximum log difference (LD). CD7, CD19, CD2, CD11b and CD56 were the most sensitive and reliable markers for MRD studies. LAPs were rarely detected in either normal or regenerating BMs. Through dilutional experiments from 50% LAPs to 0.001%, it was determined that 1 leukaemic in 104 and 105 normal cells could be detected using the improved techniques. Of the 54 patients, 31 received chemotherapy, with 27 achieving complete remission (CR). Two were LAP negative and thus 25 were evaluable for MRD post induction and 22-post consolidation chemotherapy. Detection of MRD >0.15% was able to distinguish between two groups of patients according to relapse status. Although, the number of patients was small, detection of MRD post induction > 0.15% was shown to be an independent predictor of adverse prognosis for both relapse free survival (RFS) and overall survival (OS) in a multivariate analysis [p = 0.037 and 0.026, 95% CI (1.1-20.5 and 1.2-22.2), hazard ratio 4.7 and 5.2 respectively]. Post consolidation, there was a trend for patients with higher MRD values to show shorter RFS (p = 0.06). MFC using 5-colour allows us to detect LAPs in virtually all AML patients and our preliminary results suggest the technique is a suitable approach for MRD analysis. However, 5-colour MFC is technically challenging, resource intensive, and may not be feasible in a routine diagnostic laboratory. This led us to assess whether we could identify other potential markers for LAPs. Interleukin-3 alpha receptor- chain IL-3_ (CD123) has been suggested to be a marker of leukaemic stem cells (LSC). These cells are thought to be responsible for initiating and maintaining leukaemic cell growth post chemotherapy and hence to give rise to relapse of the disease. Therefore, we analysed 34 AML patients for expression of CD123 in the blast population and defined a population containing leukaemic stem cells using the immunophenotypic markers CD123+/CD34+/CD38-. Thirty-two (94%) of AML patients expressed CD123. We then used a molecular marker to determine whether CD123 expression was confined to the LSC. Thirtynine patients were screened for the presence of FMS-like tyrosine kinase 3 - internal tandem duplication (FLT3/ITD) as the most common molecular abnormality in AML patients. Of those, 12 (31%) were FLT3/ITD positive. In seven of them, CD34+/CD38-/CD123+ and CD34+/CD38-/CD123- populations were sorted to homogeneity by Fluorescence Activated Cell Sorting (BD FACSAriaTM Cell Sorter) and tested for FLT3/ITD. In six of seven patients with FLT3/ITD positive AML, we could not detect the mutation in the CD34+/CD38-/CD123- fraction, but the mutation was detected in the CD34+/CD38-/CD123+ fraction in all seven patients. This novel finding demonstrates that, the oncogenic event occurs in CD123 positive cells, thus supporting the concept that CD123 is a marker of the LSC in CD123 positive AML. This observation suggests novel treatment approaches employing surface marker CD123-targeting antibodies may be of use in the treatment of AML. In conclusion, we demonstrate that using five-colour MFC improves LAP detection in AML and enables MRD studies using immunophenotyping to be applied to virtually all AML patients. Additionally, it increases the sensitivity of the technique for detecting LAP populations. Moreover, evaluation of MRD post induction chemotherapy is the most sensitive time point for detection of MRD, with MRD levels >0.15% predicting relapse and worse prognosis. As an alternative to using individualised LAPs specific to each patient, CD34+/CD38-/CD123+ cells may in the future serve as a better marker for MRD studies. This marker identifies the putative LSC, which is responsible for regrowth of leukaemia and relapse of the disease. Thus, instead of looking at whole “blast” population which results in huge data analysis and interpretation for the different LAPs which may have different underlying biology, it may be more informative to look at the frequency of LSC after achieving CR using CD34+/CD38-/CD123+ as the single LAP for MRD studies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317088 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Despite high remission rate in acute myeloid leukaemia (AML) after chemotherapy, relapse of the underlying disease remains a major challenge and one of the most frequent causes of treatment failure. In this study, the presence of leukaemiaassociated phenotypes (LAPs) was first studied retrospectively using our standard diagnostic protocol with 3-colour flow cytometry. LAPs were present in 54 (64%) of 84 AML patients analysed between 2002 to 2004. The presence of LAPs was correlated with failure to respond to induction chemotherapy (p <0.05) in univariate analysis. Presence of LAPs was shown to be an independent predictor for failure to respond to induction chemotherapy with a relative risk ratio of 1.6 (p < 0.05, 95% CI, 1.0-2.6) in multivariate analysis. Subsequently, in a prospective study, we used 5-colour multiparametric flow cytometry (MFC) for detection of LAPs to determine if LAPs could be detected in a greater proportion of leukaemic patients and minimal residual disease (MRD) detection could therefore be applied in more patients. In 54 consecutive, newly diagnosed AML patients from 2005 to 2007, LAPs were identified in 51 (94%). Thus, MRD studies were potentially applicable to virtually all patients. The sensitivity and specificity of MFC technique was improved by analysing 10 normal and 5 regenerating bone marrows (BM) for the presence of these LAPs and by determining maximum log difference (LD). CD7, CD19, CD2, CD11b and CD56 were the most sensitive and reliable markers for MRD studies. LAPs were rarely detected in either normal or regenerating BMs. Through dilutional experiments from 50% LAPs to 0.001%, it was determined that 1 leukaemic in 104 and 105 normal cells could be detected using the improved techniques. Of the 54 patients, 31 received chemotherapy, with 27 achieving complete remission (CR). Two were LAP negative and thus 25 were evaluable for MRD post induction and 22-post consolidation chemotherapy. Detection of MRD >0.15% was able to distinguish between two groups of patients according to relapse status. Although, the number of patients was small, detection of MRD post induction > 0.15% was shown to be an independent predictor of adverse prognosis for both relapse free survival (RFS) and overall survival (OS) in a multivariate analysis [p = 0.037 and 0.026, 95% CI (1.1-20.5 and 1.2-22.2), hazard ratio 4.7 and 5.2 respectively]. Post consolidation, there was a trend for patients with higher MRD values to show shorter RFS (p = 0.06). MFC using 5-colour allows us to detect LAPs in virtually all AML patients and our preliminary results suggest the technique is a suitable approach for MRD analysis. However, 5-colour MFC is technically challenging, resource intensive, and may not be feasible in a routine diagnostic laboratory. This led us to assess whether we could identify other potential markers for LAPs. Interleukin-3 alpha receptor- chain IL-3_ (CD123) has been suggested to be a marker of leukaemic stem cells (LSC). These cells are thought to be responsible for initiating and maintaining leukaemic cell growth post chemotherapy and hence to give rise to relapse of the disease. Therefore, we analysed 34 AML patients for expression of CD123 in the blast population and defined a population containing leukaemic stem cells using the immunophenotypic markers CD123+/CD34+/CD38-. Thirty-two (94%) of AML patients expressed CD123. We then used a molecular marker to determine whether CD123 expression was confined to the LSC. Thirtynine patients were screened for the presence of FMS-like tyrosine kinase 3 - internal tandem duplication (FLT3/ITD) as the most common molecular abnormality in AML patients. Of those, 12 (31%) were FLT3/ITD positive. In seven of them, CD34+/CD38-/CD123+ and CD34+/CD38-/CD123- populations were sorted to homogeneity by Fluorescence Activated Cell Sorting (BD FACSAriaTM Cell Sorter) and tested for FLT3/ITD. In six of seven patients with FLT3/ITD positive AML, we could not detect the mutation in the CD34+/CD38-/CD123- fraction, but the mutation was detected in the CD34+/CD38-/CD123+ fraction in all seven patients. This novel finding demonstrates that, the oncogenic event occurs in CD123 positive cells, thus supporting the concept that CD123 is a marker of the LSC in CD123 positive AML. This observation suggests novel treatment approaches employing surface marker CD123-targeting antibodies may be of use in the treatment of AML. In conclusion, we demonstrate that using five-colour MFC improves LAP detection in AML and enables MRD studies using immunophenotyping to be applied to virtually all AML patients. Additionally, it increases the sensitivity of the technique for detecting LAP populations. Moreover, evaluation of MRD post induction chemotherapy is the most sensitive time point for detection of MRD, with MRD levels >0.15% predicting relapse and worse prognosis. As an alternative to using individualised LAPs specific to each patient, CD34+/CD38-/CD123+ cells may in the future serve as a better marker for MRD studies. This marker identifies the putative LSC, which is responsible for regrowth of leukaemia and relapse of the disease. Thus, instead of looking at whole “blast” population which results in huge data analysis and interpretation for the different LAPs which may have different underlying biology, it may be more informative to look at the frequency of LSC after achieving CR using CD34+/CD38-/CD123+ as the single LAP for MRD studies. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317088 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

The 21st Century has seen a rise in incidence of complex diseases such as cancer and in the quest to develop essential new therapeutic options, the study of drug-cell interactions can yield powerful information. Acute myeloid leukaemia (AML) is an aggressive cancer that causes life-threatening deficits of functional blood cells in humans for which current treatment options are highly toxic and often poorly tolerated. A combination of two existing drugs, bezafibrate and medroxyprogesterone acetate in a drug redeployment situation has shown promise in vitro and in vivo and further investigations are crucial to elucidate the mode of action of this treatment. This project investigated the mechanistic action of BaP at a cellular level. Orthogonal spectroscopic and mass spectrometric platforms were employed to probe the biochemical composition of two AML cell lines, HL60 and K562 in the presence and absence of this combined drug treatment. Analysis was performed on single living cells, dehydrated cells, fixed cells and cell extracts to give a large and detailed data set. A consideration of the main spectral differences obtained by Synchrotron-FTIR and ATR-FTIR in conjunction with multivariate statistical analysis revealed a significant change to the cellular lipid composition with drug treatment; furthermore, this response was not caused by cell apoptosis. In particular, the ratio of CH2:CH3 was observed to increase with BaP treatment and this was determined to be a significant change in both cell lines (p &lt;0.05). An overall increase in lipid unsaturation suggests that BaP targets cellular lipid biosynthesis. Raman microspectroscopy added a further dimension to the spectroscopic study by providing spatial information of lipid distribution which suggested that BaP-induced saturation change is uniform across a single cell. UHPLC-MS was employed for global metabolomics analysis of AML cell extracts and revealed a number of biochemical pathways that were indicated as targets of BaP therapy in both cell lines. Univariate and multivariate analysis determined statistically significant metabolites for which putative identifications were made. Pyrimidine metabolism was the most significant pathway identified for changes consistent in both HL60 and K562 cell lines. The complementarity of ToF-SIMS and UHPLC-MS provided large coverage of the lipidome of AML cells through untargeted and targeted approaches. For data derived by both techniques, a general increase in polyunsaturated species for BaP treated cell extracts was observed which correlated well with findings from spectroscopic investigations. Adopting a multi-disciplinary approach to cell analysis can afford a powerful insight into understanding drug mode of action at a cellular level and novel information regarding BaP mechanistic action in AML cell lines was revealed. This analytical approach could be extended to the future study of drug-cell interactions for other oncological systems.