Project description:Acute myeloid leukaemia (AML) is an aggressive haematological malignancy characterised by the clonal proliferation of myeloid progenitor cells in the bone marrow and peripheral blood. Dysregulation of the Wnt/β-catenin pathway has been implicated in the establishment and maintenance of leukaemic stem cells in AML, where higher expression of β-catenin promotes clonogenic capacity, drug resistance and inferior survival. The finding that low levels of Wnt signalling are necessary to maintain normal haematopoiesis makes β-catenin an attractive therapeutic target, however drug design has been hampered by a poor understanding of its molecular interactions in leukaemia cells. To address this, we previously characterised the β-catenin interactome in myeloid cells and identified a plethora of novel interacting proteins. One such validated interactor was Target of EGR1 (TOE1), a 510-amino acid member of the Asp-Glu-Asp-Asp (DEDD) family of deadenylases with previously uncharacterised function in haematopoietic cells. The β-catenin:TOE1 interaction was detected in the nuclear and cytosolic compartments of myeloid cell lines and primary AML samples, and β-catenin knockdown via short hairpin RNA (shRNA) was found to promote the cytosolic stabilisation of TOE1. Furthermore, TOE1 levels were found to be overexpressed in primary AML blasts versus normal cord-blood derived CD34+ haematopoietic stem and progenitor cells (HSPCs), suggesting that TOE1 levels may be dysregulated in leukaemia. TOE1 depletion using shRNA abrogated Wnt signalling capacity (TCF/LEF activity) through suppression of the Wnt transcription factor lymphoid enhancing factor 1 (LEF-1), likely via post-transcriptional mechanisms. TOE1 shRNA further suppressed the proliferation and survival of myeloid leukaemia cell lines (HEL and OCI-AML2) and primary human CD34+ HSPC, however this could not be fully explained through LEF-1 alone, since OCI-AML2 do not express LEF-1. Therefore, to explore a more unifying mechanism mediating the growth promoting phenotype of TOE1 we performed tandem mass tag (TMT)-labelling coupled to mass spectrometry analysis in TOE1 deficient HEL and OCI-AML2 cells. From this analysis we identified validated p21 (RAC1) activated kinase 2 (PAK2) as a consistently downregulated target across both cell lines, and demonstrated PAK2 knockdown alone was sufficient to reduce the proliferation (but not survival) of AML cell lines. Finally, expression of ectopic PAK2 was able to partially rescue the suppressed proliferation mediated by TOE1 depletion in myeloid cell lines, and primary human CD34+ HSPC. In summary, these data reveal TOE1 as novel interacting partner for β-catenin in haematological cells capable of modulating Wnt signalling output via LEF-1, and as a novel mediator of growth and survival in human HSPC and AML cells partly through PAK2 regulation.

Project description:Given the importance of deregulated phosphoinositide (PI) signaling in leukemic hematopoiesis, genes coding for proteins that regulate PI metabolism may have significant and as yet unappreciated roles in leukemia. We performed a targeted knockdown screen of PI modulator genes in human AML cells and identified candidates required to sustain proliferation or prevent apoptosis. One of these, the lipid kinase phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A) regulates cellular levels of phosphatidylinositol-5-phosphate (PtsIns5P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). We found PIP4K2A to be essential for the clonogenic and leukemia-initiating potential of human AML cells, and for the clonogenic potential of murine MLL-AF9 AML cells. Importantly, PIP4K2A is also required for the clonogenic potential of primary human AML cells. Its knockdown results in accumulation of the cyclin-dependent kinase inhibitors CDKN1A and CDKN1B, G1 cell cycle arrest and apoptosis. Both CDKN1A accumulation and apoptosis were partially dependent upon activation of the mTOR pathway. Critically, however, PIP4K2A knockdown in normal hematopoietic stem and progenitor cells, both murine and human, did not adversely impact either clonogenic or multilineage differentiation potential, indicating a selective dependency which we suggest may be the consequence of the regulation of different transcriptional programmes in normal versus malignant cells. Thus, PIP4K2A is a novel candidate therapeutic target in myeloid malignancy. AIM: to determine the transcriptional consequences of Pip4ka knockdown in murine normal and leukaemic bone marrow stem and progenitor cells. KIT+ stem and progenitor cells were immunomagnetically recovered from the bone marrow of two female C57/BL6 mice, pooled and cultured in RPMI with 20% FBS supplemented with SCF 300ng/mL, FL 1ng/mL and IL-11 20ng/mL. Next day these cells were spinfected with lentiviral supernatant (shPip4k2a constructs #1, #2 or a non-targeting control). Likewise, the same viral supernatant was used to spinfect (cryopreserved, thawed and overnight cultured) leukemic blasts recovered from the BM of mice with MLL-AF9 AML (initiated using a retroviral transduction and transplantation approach). Cells were FACS purified for GFP expression (the selectable marker) 48h following lentiviral infection and returned to culture. Seventy-two hours following lentiviral infection control and Pip4k2a KD cells were recovered from culture for mRNA extraction.

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