File(s) under permanent embargo
Reason: Restricted by author. A copy can be supplied under Section 51(2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library or by emailing firstname.lastname@example.org
Characterizing the role of inositol polyphosphate 4-phosphatase type II (INPP4B) in acute myeloid leukaemia
thesisposted on 22.02.2017, 03:10 by Rijal, Sewa
Acute myeloid leukaemia (AML) is an aggressive blood cancer, fatal if not responsive to treatment. The standard induction treatment for AML, for the past three decades, involves a (7+3) chemotherapy regimen where a continuous infusion of cytosine arabinoside (ara-c) is given for 7 days in combination with an anthracycline, usually daunorubicin for 3 days [1 ]. Primary treatment failure occurs in 20-40% of adult cases, with higher rates of chemoresistance observed in elderly patients [2-4]. The activation of the phosphoinositide 3-kinase (PI3K) pathway is a feature of many cancers including acute myeloid leukaemia (AML). In fact, constitutive activation of the P13K target AKT is seen in the majority (50%-80%) of AML cases and associated with poor prognosis . The basis for activated AKT in AML remains poorly understood. Unlike in solid tumours [6, 7], activating mutations of AKT or PIK3CA have not been reported in AML [8-10]. The P13-K/Akt pathway is regulated by a triad of lipid phosphatases, known functionally as inositol polyphosphatase (INPP) enzymes, such as PTEN (3-phosphatase), SHIP1 (5-phosphatases) and INPP48 (4-phosphatases). Studies have found mutations or loss of PTEN to be absent in AML whereas mutations in SH/P-1 to account for only extremely rare cases (11-13]. There are a number of other INPP enzymes whose expression and function have not been explored in AML. We, therefore, sought to identify the presence and consequences of pathological expression of INPPs in AML. We utilized the Sequenom® MassARRAY platform to quantitatively screen the gene expression profile of key human INPP enzymes in primary AML and normal bone marrow (BM) samples. Contrary to our expectation, we identified significantly increased expression of the 4-phosphatase INPP48 in AML compared to normal BM (p=0.02). Overexpression of INPP4B in AML bone marrow (2::50% blasts positive) was confirmed in 12% of AML cases at diagnosis (n=205). INPP48 overexpression was associated with an inferior response to chemotherapy leading to a significantly shorter leukaemia-free survival (LFS) (median 6.2 vs 11.8 months; p=0.01)and overall survival (OS) (median 11.5 vs 26.6 months; p<0.01) outcomes. Multivariate analysis revealed that high INPP4B was an independent predictor of poor OS outcome for patients with AML at diagnosis (hazard ratio [HR] 2.2), along with adverse risk karyotypes (HR 1.7) and increased age (HR 2.1 ). Ectopic overexpression of INPP48 in human leukaemic cell lines conferred resistance to standard cytotoxic drugs used to treat AML, including ara-C and anthracyclines using in vitro cell death and colony-forming assays. INPP4B overexpression also led to impaired clearance of bone marrow blasts by cytarabine in vivo, as well as significantly reduced OS in human xenograft models of AML (p<0.0001 ). Although INPP48 phosphatase function was proven to be catalytically active in primary AML samples, expression of a phosphatase inactive mutant (INPP4B C842A) did not abrogate chemoresistance in vitro or in vivo. In contrast, siRNA-mediated knockdown of endogenously overexpressed INPP4B sensitized AML cell lines and primary cells to araC in vitro. Although, INPP4B has previously been reported to be a putative tumour suppressor in epithelial cancers [14-16], these findings infer 1) the presence of a novel phosphataseindependent function for INPP4B and 2) an unsuspected role for INPP48 overexpression in diminishing chemotherapy responses leading to poor survival outcome in AML. The reduction in chemoresistance associated with INPP48 inhibition highlights a potential role for this protein as a therapeutic target for future development in AML.