Abstract
Abstract 59
Identification of genes and cellular pathways that are critical for the function of leukemia stem cells (LSC) could lead to novel therapeutic approaches. We present data which demonstrates that TTC5 (also called Strap) is required in acute leukaemogenesis. In a targeted lentiviral shRNA knockdown (KD) screen of chromatin regulatory factors, we identified the EP300 co-factor TTC5 as required for survival of human THP-1 MLL-AF9 acute monoblastic leukaemia cells. TTC5 is a tetratricopeptide repeat (TPR) motif protein known to be a stress responsive co-factor and activator of the histone acetyltransferase EP300. Using two separate shRNAs targeting different parts of the transcript, TTC5 KD in THP-1 cells resulted in G1 cell cycle arrest and the initiation of myeloid differentiation (as evidenced by down regulation of KIT/CD117 and up regulation of the monocyte differentiation marker CD36 over 48–72 hours) prior to apoptosis. Colony formation in semi-solid culture was completely abolished. TTC5 KD also induced apoptosis and loss of colony forming potential in other myeloid leukaemia cell lines including Fujioka, Kasumi-1, NB4, HL60, K562 and Mono-Mac-1. In xenotransplantation assays, TTC5 KD THP-1 cells failed to initiate AML in contract to control cells infected with a non-targeting control (NTC) hairpin, which induced short latency AML (range 51–72 days). Likewise, lentivirally infected TTC5 KD primary human acute lymphoblastic leukaemia cells from a patient with a t(4;11) translocation, the cytogenetic hallmark of MLL-AF4, failed to induce lethal leukemia when xenografted into neonatal mice, in contrast to NTC cells which induced lethal ALL in recipients of median latency 117 days. Separately, TTC5 KD induced proliferative arrest, differentiation and death in in vitro analyses of primary human AML cells which exhibited MLL associated translocations or trisomy 11 (frequently associated with a partial tandem duplication of MLL) (n=4 patients). These data suggest a critical role for TTC5 in human leukaemic haematopoiesis.
To confirm our conclusions, we extended our investigations in a murine model of human leukaemic haematopoiesis initiated by MLL-AF9. TTC5 KD MLL-AF9 AML cells formed significantly fewer colonies in methylcellulose versus control cells and failed to initiate AML in secondary transplantation experiments versus control cells which induced short latency disease (48 days).
To investigate the mechanism by which TTC5 knockdown initiates the cell cycle arrest and myeloid differentiation phenotype observed in THP-1 cells, we performed exon array analyses 48 hours following lentiviral infection. Gene set enrichment analysis (GSEA) demonstrated co-ordinate down regulation of an MLL leukemia stem cell maintenance program as well as genes bound by the transcription factor MYB. In contrast there was co-ordinate up regulation of genes known to be positively regulated by PU.1 or IRF8, two myeloid transcription factors that promote differentiation. These significant transcriptional changes occurred in the absence of change in the levels of MYB or PU.1 transcripts, arguing that TTC5 may be critical in regulating the function of these transcription factors rather than modulating their expression. Published data from the FANTOM4 consortium identified MYB and, to a lesser extent, PU.1 as key transcriptional regulators of CD36 expression (Suzuki et al., 2008, Nature Genetics). We confirmed up regulation of CD36 upon MYB knockdown in THP-1 cells. CD36 up regulation was not observed when either MYC or EP300 was knocked down, suggesting that TTC5 is required for MYB function and loss of TTC5 perturbs MYB activity, initiating a differentiation program. The mechanism by which TTC5 regulates MYB activity is under investigation but appears independent of EP300. We propose that in addition to the role of TTC5 in the stress response, it is a critical cofactor protein that regulates haematopoietic transcription factors and is required for human leukaemogenesis.
Disclosures:
No relevant conflicts of interest to declare.
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