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X. Wang et al.
gest that Rheb1 may regulate neutrophil differentiation partially through mTORC1 signaling pathway. Δ/Δ To investigate the function effects of 3BDO on Rheb1 HSCs, we performed a long-term culture assay in vitro and foundΔ/Δthat the CAFC (cobblestone area) formed by Rheb1 HSCs was similiar to that of the control in both 3BDO and DMSO groups (Online Supplementary Figure S6A and B).We further treated Rheb1Δ/Δ and Rheb1fl/fl BM cells with 3BDO or DMSO. Then, we transplanted Rheb1Δ/Δ and Rheb1fl/fl BM cells (CD45.1) together with com- petitive cells (CD45.2) to lethally irradiated mice. The chimerism was analyzed every four weeks after transplan- tation. We found that the repopulating capacity of Rheb1Δ/Δ BM cells in the PB was significantly lower when compared with Rheb1fl/fl cells, both in 3BDO and DMSO groups (Online Supplementary Figure S6C and D). These data indi- cated that Rheb1 may regulate HSCs engraftment inde-
pendently of the mTORC1 signaling pathway.
Discussion
Although Rheb1 has been known to regulate TSC1/2 upstream of mTORC1, its role in the regulation of hematopoiesis is still not fully understood. The somatic loss-function mutation in the RHEB gene in AML patients also provides genetic evidence that mutational inactivation of RHEB might be a pathogenic event in myeloid malignan- cies (Figure 5). Notably, we showed here that AML patients with low RHEB expression had shorter survival time than AML patients with high RHEB expression, indicating that RHEB could be a prognosticator for leukemia patient sur- vival. In a Rheb1 conditional deletion mouse model, we determined that loss of Rheb1 caused defective HSCs and an increased number of immature neutrophils in BM, accompanied by excessive extramedullary hematopoiesis in the spleen. In addition, Rheb1 loss leads to progressive myeloproliferation in aged Rheb1Δ/Δ mice. These data sug- gest that Rheb1 participates in proliferation and differentia- tion in myeloproliferative disease.21 Our gene transcriptional expression data also reinforce the idea that Rheb1 loss leads to an increased expression of myeloid leukemia-related gene expression programs (Figure 6A and B). Further studies are needed to functionally dissect the downstream targets of Rheb1 that confer enhanced proliferation ability for stem/progenitor cells or LSCs.
In the steady-state condition, the majority of HSCs are in a quiescent state.22 However, when mice are under hematopoietic stresses, such as transplantation, HSCs actively proliferate to generate progenitors that enable rapid hematologic regeneration.23 Successful reconstitution upon transplantation of HSCs depends on multiple param- eters, including correct homing to the BM, residing and proliferating successfully in the BM niche, the right cell- cycle status of the transplanted cells, and the adequate rate of apoptosis. Here, although Rheb1Δ/Δ mice could survive with increased HSCs and HPCs in BM and spleen to com- pensate for the loss of Rheb1 under steady-state condi- tions, Rheb1Δ/Δ HSCs were unable to reconstitute adult BM in transplantation. However, CAFC assay showed the pro- liferation capacity of Rheb1Δ/Δ HSCs was similar to that of Rheb1fl/fl HSCs in vitro (Online Supplementary Figure S6A and B). Our results thus suggested that the overproliferation of HSCs in Rheb1Δ/Δ mice may be caused by multiple factors including extrinsic factors such as BM microenvironment.
Indeed, our gene transcriptional expression data showed that Rheb1 loss leads to a decrease in expression of adhe- sion-related gene expression programs (Figure 6A and B). Rheb1 may regulate HSC regeneration through non- canonical signaling pathways rather than being fully dependent on mTORC1 signaling pathway. This is consis- tant with the findings of Peng et al. that the proliferation ability of HSCs was impaired in Raptor-deficient HSCs (Raptor is a component of mTORC1 downstream of Rheb1), but not in Rheb1-deficient HSCs upon transplan- tation.24 Interstingly, in Peng et al.’s model of TAM injection at eight weeks post transplantation of Rheb1fl/fl ;Rosa- CreERT2 BM cells into wild-type (WT) recipient mice, no significant difference was observed in the regeneration of donor cells when compared to WT competitor cells. This is also consistant with the idea that Rheb1 deficiency does not affect HSC proliferation.24 It will be important to deter- mine the contribution of Rheb in canonical and non-canon- ical signaling pathways in future studies.
Neutrophils are specially developed cells to provide a defense against bacterial infection and are essential for host survival. Rheb1Δ/Δ mice showed severe neutrophilia in PB with an increased percentage of immature neutrophils in the BM. The augmented GMP/CMPs may be the reason for the neutrophilia, as the absolute number of GMP/CMPs significantly increased in the BM and spleen of Rheb1Δ/Δ mice (Figures 1B and 2F). Although the number of neutrophils was higher in the PB and BM in Rheb1Δ/Δ mice when com- pared to the control (Online Supplementary Figure S1A), Rheb1Δ/Δ neutrophils could not kill bacteria effectively in vitro (Figure 1J). It has been reported that Raptor deficiency resulted in decreased numbers of Gr-1+Mac-1+ (Gr- 1+CD11b+) granulocytes.25 However, our results showed that Rheb1 deletion led to an increase in the number of Ly- 6G+CD11b+ granulocytes. This discrepancy may be due to Ly-6G marking only a subset of Gr-1 cells, while Ly-6G is used for separating granulocytes from Mac-1+ myeloid cells. Furthermore, we found mTORC1 signaling was inhibited in Rheb1Δ/Δ progenitor cells, as evidenced by a reduced level of p-S6 (Figure 6C and D). A specific mTORC1 activator, 3BDO could only partially rescue Rheb1 deficiency- induced immature neutrophils, indicating Rheb1 regulates neutrophil development at least partially via the mTORC1 pathway. It is possible that Rheb1 regulates neutrophil dif- ferentiation via other signaling pathways. This possibility still needs to be explored in future studies.
We have shown that mutations or reduced expression of RHEB are associated with leukemia. Interestingly, although Rheb1 deficiency leads to an MPN-like disorder in aged Rheb1Δ/Δ mice, loss of Rheb1 shortens the life of Rheb1Δ/Δ mice but does not lead to spontaneous leukemia in Rheb1Δ/Δ mice in our observations (Figures 4 and 5). It is possible that the reduced survival of aged Rheb1Δ/Δ mice may be due to ineffective myelopoiesis-related inflammation. Although Rheb1-deficiency was not a key factor for leukemogenesis, low expression of Rheb1 does associate with the initiation of myeloid proliferation-related diseases, such as MPN. We predict that additional mutations and/or pro-leukemia development environment factors are needed to initiate leukemogenesis in Rheb1Δ/Δ hematopoietic cells.
Acknowledgments
The authors thank Dr. Bo Xiao for providing the Rheb1fl/fl mice, Dr. Junying Miao for providing the 3BDO reagent, and Ms. Xiaohuan Mu and Yuemin Gong for technical assistance.
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haematologica | 2019; 104(2)