Acute myeloid leukemia (AML) is a life-threatening malignant disorder with dismal prognosis. AML is characterized by frequent genetic changes involving tyrosine kinases, normally acting as important mediators in many basic cellular processes. Due to the overexpression and frequent mutations of the FMS-like receptor tyrosine kinase 3 (FLT3) in AML, this tyrosine kinase receptor has become one of the most sought after targets in AML drug development. In this thesis, we have used a combination of high-throughput screens, direct target interaction assays and sequential cellular screens, including primary patient samples, as an approach to discover new targeted therapies. Gefitinib, a previously known inhibitor of epidermal growth factor receptor and the two novel tyrosine kinase inhibitors AKN-032 and AKN-028, have been identified as compounds with cytotoxic activity in AML. AKN-028 is a potent inhibitor of FLT3 with an IC50 value of 6 nM in an enzyme assay, but also displaying in vitro activity in a variety of primary AML samples, irrespective of FLT3 mutation status or quantitative FLT3 expression. AKN-028 shows a sequence dependent in vitro synergy when combined with standard cytotoxic agents cytarabine or daunorubicin, with better efficacy when cells are exposed to standard chemotherapy simultaneously or for 24 hours prior to adding AKN-028. Antagonism is observed when cells are pre-treated with AKN-028, possibly explained by the cell cycle arrest induced by the compound. In vivo cytotoxic activity and good oral bioavailability have made AKN-028 a candidate drug for clinical studies and the compound is presently investigated in an international two-part multicenter phase I/II study. Results from microarray studies performed to further elucidate the mechanism of action of AKN-028, revealed significantly altered gene expression induced by AKN-028 in both AML cell lines and in primary AML cells, with an enrichment of the Myc pathway among the downregulated genes. Furthermore, tyrosine kinase activity profiling shows a dose-dependent kinase inhibition by AKN-028 in all AML samples tested. Interestingly, cells with a high overall kinase activity were more sensitive to AKN-028. Provided conformation in a larger set of samples, kinase activity profiling may give useful information in individualizing treatment of patients with AML.

Around 50% of chronic myeloid leukaemia (CML) patients who remain on imatinib treatment for more than 5 years will achieve a complete molecular response (CMR), defined by undetectable BCR-ABL mRNA in a sensitive reverse transcriptase real-time quantitative PCR (RQ-PCR) assay. Given the increasing importance of CMR on imatinib therapy the primary aim of this study was to improve the accuracy and sensitivity of MRD detection to allow a more accurate estimation of relapse risk when therapy is withdrawn. Firstly, we investigated ways of improving the sensitivity of RT-PCR methods for the detection of BCR-ABL mRNA. Secondly, we investigated the use of the patient-specific BCR-ABL gene for the detection of MRD. Thirdly, we conducted a multi-centre clinical trial of imatinib withdrawal in selected CML patients in a stable CMR. This clinical trial provided patient samples that could be used to test our optimized MRD assays, and provided clinical data on the risk and patterns of relapse after withdrawal of imatinib therapy. The trial is ongoing, but an interim analysis of the study data was performed. In 22 patients the estimated probability of molecular relapse after imatinib withdrawal was 54%, and 60% of relapses occurred within the first 4 months. The average detection limit of BCR-ABL mRNA by RQ-PCR is estimated at around 4.5 log below the level of BCR-ABL prior to commencing treatment. The number of leukaemic cells at diagnosis is around 10¹ ², so the number of residual leukaemic cells in CMR might vary from zero to over a million. We hypothesized that the amount of residual leukaemia in CMR is variable between patients, and that this heterogeneity is a determinant of the risk of relapse when treatment is withdrawn. We developed more sensitive methods for the detection of BCR-ABL and tested these methods in samples from our study patients. We showed that random pentadecamer (15-mer) primers improved the efficiency of reverse transcriptase PCR (RT-PCR), and resulted in a lower detection limit of BCR-ABL mRNA. We also developed a novel nested RT-PCR method using real-time PCR for the second round of the reaction, and this resulted in a lower detection limit of BCR-ABL in patient samples. The utility of this nested RT-PCR method was limited by a false positive rate of 2-3% in the HeLa cell line that we used as our negative control. Consequently, we examined the detection of the patient-specific genomic BCR-ABL sequence as an alternative to RT-PCR. Breakpoints in BCR and ABL1 in CML patients are widely dispersed over 3 kb and 150 kb, respectively. Therefore, the BCR-ABL genomic sequence is essentially unique to each patient. We sequenced the genomic breakpoints of 43 CML patients. We showed that the distribution of breakpoints in BCR and ABL1 was non-random, but we were unable to identify any genomic feature that determined the specific location of individual breakpoints. We developed a novel BCR-ABL DNA Q-PCR method for 12 of the study patients, and in 11 of the patients BCR-ABL DNA was detected when the patient was in a CMR, confirming that this method was more sensitive than RQ-PCR. Contrary to our hypothesis, the detection of BCR-ABL DNA was not predictive of relapse. In most patients who relapsed there was a significant increase in BCR-ABL DNA prior to mRNA relapse. Two patients had stable levels of BCR-ABL DNA measurable on multiple occasions, but remained in remission after 6 months and 15 months, respectively. We have shown that a stable CMR after the withdrawal of imatinib therapy does not necessarily indicate the eradication of leukaemia. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2010

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001. / "December 2001." Includes bibliographical references (leaves 178-225).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 13, 2008). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 14, 2010). Includes bibliographical references.

Thesis--University of Oklahoma. / Bibliography: leaves 90-93.

Tumor-infiltrating myeloid-derived suppressor cells (MDSCs), are important mediators of a tumor-permissive microenvironment that contributes to tumor growth and could account for the limited success of immunotherapeutic strategies. MDSCs suppress adaptive immunity by blocking T cell activation, inducing Treg accumulation, and inhibiting natural killer cell cytotoxicity against tumor cells. We investigated the roles of MDSCs in the regeneration of the exocrine pancreas associated with acute pancreatitis and the progression of acinar to ductal metaplasia. Acute pancreatitis was induced in wild type and P48+/Cre;LSL-KRASG12D mice using caerulein and an early influx of MDSCs into the pancreas was observed flow cytometry and immunocytochemistry. Numbers of Gr1(+)CD11b(+) MDSCs increased over 20-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes and have T cell suppressive properties. This marked accumulation of MDSCs returned to normal values within 24 hours of the insult in wild type mice; however, in the oncogenic KRAS mice, MDSCs levels remained elevated. When intrapancreatic MDSCs were depleted by administration of a CXCR2 antagonist (SB265610) in wild type mice the severity of acinar damage was increased. This was also accompanied by a delayed regeneration determined morphologically and with the mitotic immunomarker phospho-histone H3. Isolated intrapancreatic MDSCs from treated mice induce naïve acinar cells to undergo acinar ductal metaplasia when co-cultured in collagen 3D cultures. Purified splenic MDSCs failed to induce the phenotypic transdifferentiation. We conclude that MDSCs are required for adequate pancreatic regeneration in wild type mice with acute pancreatitis and their persistent elevation in oncogenic KRAS mice is not only associated with immune-evasion, but may also function as direct enhancer of malignant proliferation.

Acute myeloid leukaemia (AML) and ovarian cancer (OVC) are two difficult to treat cancers. AML is often treatable however minimal residual disease (MRD) endures such that many patients who achieve remission eventually relapse and succumb to the disease. OVC affects approximately 7000 women in the U.K. every year. It can occur at any age but is most common after menopause. Diagnosis at an early stage of disease greatly improves the chances of survival however, patients tend to be diagnosed in the later stages of disease when treatment is often less effective. Immunotherapy has the potential to reduce MRD and delay or prevent relapse. In order for immunotherapy to work, tumour antigens need to be identified and characterised so they can be effectively targeted. Personalised treatments require the identification of biomarkers, for disease detection and confirmation, as well as to provide an indication of best treatment and the prediction of survival. PASD1 has been found to be frequently expressed in haematological malignancies and I wanted to determine if there was a correlation between the presence of antigen-specific T cells in the periphery of patients with AML and PASD1 protein expression in the leukaemic cells. The expression of other leukaemia antigens were concurrently examined as comparators. I performed RT-PCR on nine antigens and immunocytochemistry on PASD1 in 18 samples from AML patients. I found a correlation between PASD1 expression in AML samples and the presence of PASD1-specific T cells as detected on the pMHC array. OVC lacks suitable targets for immunotherapy with few CTAs having been identified. I examined the expression of SSX2IP and the CTAs PASD1 and SSX2 in OVC. I compared the protein expression of these known tumour antigens to the “gold standard” biomarker for the diagnosis of OVC, CA125 and two other proteins known to be promising in the diagnosis of OVC, HE4 and WT1. I analysed commercially available paraffin-embedded OVC multiple tissue arrays (MTAs) containing 191 samples, predominantly stage I (n= 166), II (n= 15) and III (n= 6) OVC as well as healthy donor (n= 8) and normal adjacent tissues (n= 8). Scoring was performed in a single blinded fashion. I found SSX2A to be expressed at a score level of 3 with a frequency (37/191) that exceeded that of CA125 (14/191), HE4 (14/191), WT1 (1//191) or PASD1 (0/191). To confirm this expression I used two additional commercially-available antibodies that recognise the region common to SSX2A and B, and an antibody specific for SSX2A. Using SSX2 peptides, I blocked the immunolabelling of SSX2 in SSX2-positive cell lines showing that the immunolabelling of SSX2 and SSX2A was specific. I demonstrated that the expression of SSX2 and specifically SSX2A was reproducible and restricted to ovarian cancer with little or no expression in endometrial tissues, or diseased or inflamed endometrial tissue. In summary, these studies demonstrated that PASD1 expression in leukaemia cells correlated with the presence of PASD1-specific T cells in the periphery of presentation AML patients. I have shown that PASD1 specific-T cells are present in AML patients at diagnosis and that immunotherapy targeting PASD1 could be used to break tolerance and clear residual leukaemia cells during first remission. Analysis of the expression of three antigens in OVC, identified the specific expression of SSX2, in particular SSX2A in OVC but not healthy or diseased endometrial tissues. The expression of SSX2A was more frequent and more specific to OVC, than HE4 and WT1, and more frequent at higher intensity, especially in early stage OVC, than CA125. SSX2 and explicitly SSX2A requires further investigation to determine whether the high level of background at score 2 can be reduced with better blocking of non-specific sites. This may require the use of different SSX2 antibodies or an improved staining protocol.