Abstract
Mechanistic target of rapamycin (mTOR) functions within a complex signalling cascade, through its activity in two unique complexes mTORC1 and mTORC2, to promote a multitude of different cellular functions including autophagy, protein synthesis and survival. The exact role of these complexes during leukaemia initiation/maintenance remains to be elucidated. Here, using transgenic knockout (KO) mouse models, we determine the individual roles of mTORC1 (targeting raptor) and mTORC2 (targeting rictor) in normal haemopoiesis and in CLL initiation/maintenance.
Our results demonstrate that mice carrying a targeted KO of raptor at the haemopoietic stem cell (HSC) stage (Vav-cre+Raptorfl/fl ) do not survive post birth. This is due to anaemia resulting from a significant decrease in Ter119+ population, a significant decrease in KLF1 and KLF2 gene expression, and a significant increase in the megakaryocyte-erythroid population (MEP), suggesting a block at the MEP stage in Vav-cre+Raptorfl/fl foetal liver. While mTORC1 plays a fundamental role in RBC development, we show that mTORC2 plays a role in RBC regulation, as Rictor-deficient HSPCs exhibit an increase in RBC colony formation ex vivo. Conditional KO (cKO) of Raptor (Mx1-cre+Raptorfl/fl) in adult mice results in splenomegaly accompanied by increased spleen organ cellularity. There is a significant decrease in the B cell lineage, with a block in B cell development at the Lin-Sca-1+CD117+ (LSK) stage in the BM. mTORC2, on the other hand regulates late B cell maintenance as indicated by a significant decrease in transitional B cells (T1/T2), marginal zone progenitor (MZP), and follicular 1 (Fol1) cells in Vav-cre+Rictorfl/fl mice compared to controls.
To address the role of mTORC1 and mTORC2 in CLL initiation/maintenance in vitro, BM-derived haemopoietic progenitors isolated from control (Cre-), Raptor-deficient (Mx1-cre+Raptorfl/fl) or Rictor-deficient (Vav-cre+Rictorfl/fl) mice were retrovirally-transduced with a kinase dead PKCα (PKCα-KR) construct to induce an aggressive CLL-like disease. Raptor-deficient BM progenitors exhibited reduced proliferation and failed to generate a CLL-like disease, due to a block in B cell lineage commitment. However, there was an increase in cell cycling and migration in PKCα-KR CLL-like cells with Rictor- deficiency suggesting a role of mTORC2 in disease maintenance.
To determine a role for mTORC1 in disease maintenance in vivo, NSG mice were transplanted with PKCα-KR-transduced BM-isolated from either Mx1-cre-Raptorfl/fl or Mx1-cre+Raptorfl/fl. Once disease was established in vivo, cKO was induced and disease load and progression was monitored. Our data indicate a significant decrease in disease load with Raptor cKO, together with a trend towards increased survival. Ongoing experiments with Mx1-cre+Rictorfl/fl mice will give us an insight into the role of mTORC2 in CLL.
Taken together, mTORC1 plays an essential role in haemopoiesis, with Raptor-deficiency causing a block in RBC and B cell development at the MEP and LSK stage respectively. In comparison, Rictor-deficiency regulates later B cell lineages and promotes RBC colony formation, potentially through mTORC1 activation. Importantly, CLL-like cells lacking mTORC2 have increased cell cycling and migration whereas mTORC1 deficiency causes a decrease in disease load. Therefore, mTORC1 and mTORC2 play complementary roles in haemopoietic development and leukaemia initiation/progression. These studies provide a strong foundation for further studies testing clinical mTOR inhibitors for CLL in our models.
Disclosures
No relevant conflicts of interest to declare.
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