Rheb1 promotes tumor progression through mTORC1 in MLL-AF9-initiated murine acute myeloid leukemia

As the most common acute leukemia in adults, acute myeloid leukemia (AML) is a genetically heterogeneous neoplasm. Although this disease can initially be contained using classical chemotherapy, AML frequently relapses during the course of the disease [1]. The leukemia cells can be influenced by microenvironmental cues and also affect the normal niche of hematopoietic stem cells (HSCs) [2, 3]. A small fraction of AML cells can self-renew and are thus referred to as leukemic stem cells (LSCs). LSCs share properties with normal HSCs in sustaining normal hematopoietic hierarchy [4, 5]. LSCs have been implicated in the relapse of leukemia and are extensively studied as potential therapeutic targets. The signaling pathways that regulate the development and survival of these cells are of particular interest [5, 6].

Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine (Ser/Thr) kinase that senses and responds to multiple signals in eukaryotes. mTOR interacts with multi-proteins to form two distinct complexes, designated mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 primarily comprises mTOR, raptor, PRAS40, deptor, the Tti/Tel complex, and mLST8. This complex directly phosphorylates p70 ribosomal protein S6 kinase 1 (p70S6K1, phosphorylates ribosomal protein S6) and eukaryote translation initiation factor 4E binding protein 1 (4E-BP1) to regulate cell growth and survival [7]. Upon growth factor/cytokine stimulation, phosphatidylinositol 3-kinase (PI3K) is rapidly activated and recruits protein kinase B (Akt, PKB) to the plasma membrane. Akt is subsequently phosphorylated at its T308 and S473 sites. Following the phosphorylation of Akt or ERK1/2, TSC1/TSC2 is destabilized and the inhibition of mTOR signaling is relieved. The activation of the PI3K/Akt/mTOR signaling pathway has frequently been reported in AMLs [8, 9]. For example, a previous study showed that TSC1 deficiency disrupts the quiescence and long-term reconstitution of HSCs, reflecting over-activated mTORC1 [10]. The deletion of phosphatase and tensin homologue (PTEN), which negatively regulates the PI3K/Akt/mTOR pathway, leads to leukemia, including AML in mice [11]. In addition, the deletion of raptor, an essential component of mTORC1, suppresses AML progression through the enhanced apoptosis of AML cells [12]. Rapamycin, a natural mTOR kinase inhibitor, inhibits not only the generation but also the maintenance of leukemia in PTEN-evoked leukemogenesis [11]. Human AMLs have also been shown to respond to rapamycin [13], but with low efficacy, and new homologues of rapamycin (rapalogs) are being developed to treat AMLs [14].

Ras homologue enriched in the brain (Rheb) is an activator of mTORC1 [15, 16]. Two Rheb family members, Rheb1 (the original Rheb, hereafter referred to as Rheb1) and Rheb2 (also known as RhebL1), have been identified in mammals. Rheb1 is essential for murine development, as the loss of Rheb1 results in embryonic death at approximately midgestation [17], while Rheb2 is dispensable in mouse development [18]. The TSC complex, comprising TSC1, TSC2, and TBC1D7, are GTPase-activating proteins (GAPs) of Rheb [19‐21]. Active Rheb might indirectly inhibit PI3K and mTORC2 signaling through the induction of various mTORC1-dependent negative feedback loops [22]. Previous studies have demonstrated that Rheb1 was overexpressed in various cancers, such as non-small cell lung cancer, liver cancer, bladder cancer, and prostate cancer [23‐26]. The high level of Rheb1 was associated with the progression, metastasis, drug resistance, and poor prognosis of breast cancer patients [27]. The human cancer genome data analysis revealed recurrent mutations in Rheb1 [28]. In addition, Rheb1 overexpression also favors myc-mediated lymphomagenesis in a mouse model [29]. The findings indicate that Rheb1 contributes to tumorigenesis and might be a potential treatment target.

Although it has been clearly established that the dysregulation of mTORC1 promotes leukemogenesis, the role of Rheb1 in the development and maintenance of AML is not clear. In the present study, we examined the role of Rheb1 in the MLL-AF9 murine AML model and showed that Rheb1 deletion suppressed AML progression by impairing LSC function and reducing mTORC1 signaling.

Previous studies using MLL-AF9 AML models have established that LSCs are enriched in c-Kit⁺Gr-1⁻ (K⁺G⁻) [6] or L-GMP populations [32]. To further delineate AML progression without Rheb1, the differentiation status of Rheb1 ^fl/fl or Rheb1 ^Δ/Δ AML cells was analyzed using the cell surface markers, c-Kit and Gr-1. We observed that the percentage of K⁺G⁻ cells was slightly increased, whereas the percentage of K⁺G⁺ cells was decreased, and the percentage of K⁻G⁺ cells was not different in the BM of Rheb1 ^Δ/Δ GFP⁺ cells compared with the control (Fig. 3a). Although the absolute cell number of K⁺G⁻ and K⁻G⁺ cells was not increased, the number of K⁺G⁺ cells in the BM was dramatically decreased in Rheb1-deficient AML cells (Fig. 3b). The L-GMP (Lin^low GFP⁺ c-Kit⁺ FcR-γ⁺ CD34⁺) frequency was subsequently analyzed, and no difference in the L-GMP frequency was observed between the two groups (Additional file 1: Figure S2F and Fig. 3c), consistent with the frequency of K⁺G⁻ cells (Fig. 3b). In addition, the percentages of L-GMP and K⁺G⁻ cells in the spleen were also not changed between the two groups (Additional file 1: Figure S2G and H). These results indicate that the Rheb1 deletion did not affect the absolute number of LSCs in the BM.

To determine whether the prolonged survival of Rheb1 ^Δ/Δ AML mice was due to impaired homing ability of AML cells, Rheb1 ^fl/fl or Rheb1 ^Δ/Δ GFP⁺ cells were transplanted into lethally irradiated recipient mice. The recipient mice were sacrificed at 24 h after transplantation, and the BM and spleen were analyzed for GFP⁺ AML cells. The absolute number of GFP⁺ cells homing to the BM and spleen was not changed between the two groups (Additional file 1: Figure S3A), indicating that homing is not a factor underlying the prolonged survival of Rheb1 ^Δ/Δ AML mice.

To explore the transcriptional landscape changes that distinguish Rheb1 ^fl/fl or Rheb1 ^Δ/Δ AML cells, gene expression microarray was performed with sorted Rheb1 ^fl/fl or Rheb1 ^Δ/Δ GFP⁺ cells. The deletion of Rheb1 in sorted Rheb1 ^Δ/Δ GFP⁺ cells was confirmed after measuring the mRNA expression levels in these cells using RT-PCR (data not shown). Gene set enrichment analysis (GSEA) [33] revealed the significant enrichment of downregulated genes in hematopoietic precursor cells expressing HOXA9 and MEIS1, CD34⁺ hematopoietic cells expressing the NUP98-HOXA9 fusion (Fig. 3d), and CD133⁺ hematopoietic stem cells (as an alternative for CD34⁺ cells) (Fig. 3e). The enriched and selected genes were further validated by RT-PCR. HOXA9, which is required for the survival of MLL-arranged acute leukemia, is overexpressed in approximately 50 % of AML and highly associated with poor prognosis [34, 35]. Here, we observed that the HOXA9 expression level decreased due to Rheb1 deletion (Fig. 3f). The mRNA expressions of TGFβ1, MMP8 (matrix metalloproteinase 8), and LTF (lactoferrin), which all show tumor suppressing ability [36‐38], were significantly increased in Rheb1 ^Δ/Δ GFP⁺ cells compared with the control (Fig. 3f). This result suggests that Rheb1 deletion contributes to AML progression through the downregulation of HOXA9 expression and the upregulation of tumor suppressor gene expression, which potentially affects LSC function. To further investigate the effect of Rheb1 deletion on LSC function, we examined the colony-forming ability of Rheb1 ^fl/fl and Rheb1 ^Δ/Δ AML cells to characterize the progression and stemness of AML cells. The sorted GFP⁺ or K⁺G⁻ cells were cultured in methylcellulose medium supplemented with IL-3, IL-6, SCF, and GM-CSF, and the colonies were counted after 7 to 8 days of culture. A previous study established that colonies show progression in AML cell maturation from type A to type B to type C, demonstrating a progressive loss of stemness [39]. Here, we observed that the number of total colonies was significantly decreased in Rheb1 ^Δ/Δ GFP⁺ cells compared with the control. Interestingly, the number of type A colonies was reduced, while the number of type B and type C colonies was increased (Fig. 3g). Similar observations were obtained with Rheb1 ^Δ/Δ K⁺G⁻ cells compared with the control (Fig. 3h). Furthermore, K⁺G⁻ cell transplantation showed that the survival of Rheb1 ^Δ/Δ AML mice was significantly prolonged than the control mice (Fig. 3i). These results supported that the loss of Rheb1 impaired AML progression and affected the stemness of AML cells.

Rheb1 has been demonstrated as a molecular link between upstream PI3K/Akt signaling and downstream mTOR kinase to regulate cell growth [16, 47]. The PI3K/Akt/mTOR signaling pathway has been demonstrated to play numerous vital roles in cell survival and cell metabolism [48, 49]. The constitutive activation of PI3K/Akt/mTOR signaling was observed in 50–80 % of AML patients and has been associated with poor prognosis [50, 51]. Many inhibitors targeting this signaling pathway, either alone or in combination, have been developed, but with mediocre anti-leukemic efficacy [52]. Although Rheb1 has been shown to be mutated in cancer [28], the role of Rheb1 in AML remains unexplored. Here, we observed that in human AMLs, Rheb1, and mTOR mRNA were overexpressed (Fig. 1a and Additional file 1: Figure S4A). Using a Rheb1-deficient MLL-AF9 murine leukemia model, we further demonstrated that Rheb1 positively regulates leukemic cell growth via mTORC1 (Fig. 2b).

LSCs are composed of a minor subset of AML cells that are responsible for leukemia initiation, progression, and relapse [53]. LSCs are frequently insensitive to chemotherapy and therefore considered potential therapeutic targets for the eradication of cancer [54]. In the present study, the Rheb1 deletion did not change the LSC number in mouse BM, but the lifespan of AML mice was significantly prolonged. Additional experiments revealed that more Rheb1-deficient AML cells were arrested in the G0 phase with several upregulated CKIs. GSEA showed the enrichment of downregulated genes in hematopoietic progenitor or stem cells in Rheb1 ^Δ/Δ GFP⁺ cells, and some of these selected genes were confirmed by RT-PCR (Fig. 3d–f). In addition, Rheb1 ^Δ/Δ LSCs showed reduced colony formation, and the colonies exhibited reduced stemness in vitro (Fig. 3g, h). Furthermore, LSC transplantation showed Rheb1 deletion significantly prolonged the survival of AML mice in vivo (Fig. 3i). Thus, Rheb1 is required for LSC functional maintenance and AML progression. LSCs are known to resemble HSCs that give rise to all lineages of blood cells [55]. Interestingly, we found that under steady condition, Rheb1 deletion in hematopoietic system impaired HSC self-renewal and differentiation capability (data not shown) as in Rheb1 ^Δ/Δ LSCs.

Apoptosis is considered a key process for cancer progression or remission. Although apoptotic signaling circuitry in mammals is complicated and redundant with multiple regulators and effectors, this process offers attractive novel therapeutic targets along the pathway [40]. Here, we showed that the Rheb1 deletion increased the proportion of apoptotic AML cells, including LSCs, coincident with the upregulation of p53 and Bax mRNA expression (Fig. 4g–j). The enhanced apoptosis of Rheb1 ^Δ/Δ LSCs was further confirmed based on the observation that GFP⁺ cells and LSCs showed reduced p-4E-BP1, indicating the induction of apoptosis in tumor cells [56] and suggesting that Rheb1 might be a potential drug target for LSCs.

As a direct downstream effector of Rheb1, mTORC1 is essential for cell growth and metabolism. The deletion of raptor, a core component of mTORC1 [57], in the MLL-AF9 murine leukemia model, inhibited mTORC1 activity as evidenced by the decreased phosphorylation of S6 and 4E-BP1, decreased proliferation, and enhanced apoptosis of AML cells. These resulted in the prolonged survival of raptor-deficient AML mice, although the LSC number was increased in the raptor-deficient AML mouse model [12]. The discrepancy in LSC numbers in Rheb1- and raptor-deficient AML mouse models likely reflects the extent of mTORC1 inhibition. Indeed, we found that rapamycin treatment further decreased the phosphorylation level of S6 and 4E-BP1 and enhanced apoptosis in Rheb1 ^Δ/Δ GFP⁺ cells (Fig. 5e, f), while mTOR activator 3BDO increased the mTORC1 signal, partially rescued the augmented apoptosis of Rheb1 ^Δ/Δ GFP⁺ cells (Fig. 5g, h). Additionally, microarray analysis also showed that several mTORC1-independent pathways were also significantly altered (Additional file 1: Figure S4C and D). All these suggest the involvement of either a non-canonical Rheb1 pathway or the indirect effects resulting from the loss of Rheb1 [58, 59].

Despite advances in the current understanding of AML, the development of clinically effective therapies to eradicate AMLs has shown limited success, likely reflecting the existence of LSCs. Here, we convincingly showed that rapamycin could further enhance apoptosis in Rheb-deficient AML or LSC cells, suggesting the combinational use of Rheb1-specific inhibitors and improved rapalogs that better target mTORC1 might eradicate LSCs in AML patients with higher Rheb1 or mTORC1 levels.

Rheb1 is overexpressed in human AML. Rheb1 deletion prolongs the survival of AML mice by reducing mTORC1 signaling and increasing apoptosis in AML cells. Rapamycin could further reduce mTORC1 signaling and increase the apoptosis of Rheb1 ^Δ/Δ GFP⁺ cells, while 3BDO partially rescues the apoptosis of Rheb1 ^Δ/Δ GFP⁺ cells through the activation of mTORC1 pathway (Fig. 5i). Our study thus suggests that combinational therapy targeting both Rheb1 and mTORC1 might be more efficient in eradicating leukemia.