TTC5, a TPR Motif co-factor required for myeloid leukaemogenesis


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James Lynch1, Tim Somervaille1
1Cancer Research UK, Leukaemia Biology Laboratory, Paterson Institute for Cancer Research, The University of Manchester, Manchester, United Kingdom

Background

Despite recent advances in the treatment of acute myeloid leukaemia (AML), overall survival rates remain poor. Identification of genes belonging to critical cellular pathways in AML could lead to novel therapeutic approaches.

Method

Using a lentiviral shRNA knockdown screening strategy, the p300 co-factor TTC5 (Strap) was identified as a candidate survival factor in human myeloid leukaemia cells. TTC5 is a tetratricopeptide repeat (TPR) motif protein that interacts with p300 and JMY to block MDM2 mediated p53 degradation.

Results

In in vitro analyses, knockdown of TTC5 in THP-1 human MLL-AF9 monoblastic leukaemia cells induced G1 cell cycle arrest, down regulation of early haematopoietic cell surface markers CD34 and CD117, and subsequent apoptosis 96 hours following lentiviral infection. The colony forming potential in semi-solid culture of THP1 cells was also completely abrogated. Similar phenotypes were observed in other myeloid leukaemia cell lines, including Fujioka, Kasumi-1, NB4, HL60, K562 and Mono-Mac-1.

To determine whether TTC5 knockdown adversely affected leukaemia-initiating cell potential, TTC5 KD or control THP-1 cells were injected into NSG mice in xenogeneic transplantation assays. Mice injected with 10,000 control KD cells died of leukaemia with a median latency of 81 days (range 75-97), whereas all the mice injected with 10,000 viable TTC5 KD cells survived for 180 days, and at post-mortem analysis lacked any evidence of human leukaemia cell engraftment.

In experiments using primary human AML cells, TTC5 KD similarly induced cell cycle arrest in in vitro cultured cells but additionally promoted terminal myeloid differentiation to mature macrophages.

In THP-1 TTC5 KD cells the level of p53 protein does not change significantly, whereas p21 is induced at both mRNA and protein level, suggesting a previously unknown mechanism of action and role for TTC5 in myeloid leukaemogenesis.

Conclusion

These results demonstrate that the cofactor TTC5 is required for proliferation and survival of myeloid leukaemia cells.