A targeted knockdown screen of genes coding for phosphoinositide modulators identifies PIP4K2A as required for acute myeloid leukemia cell proliferation and survival

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Tim Somervaille 

PROVIDER: E-GEOD-54309 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress