Hes1 expression and CYLD repression are essential events downstream of Notch1 in T-cell leukemia

Abstract

Notch activation is a current event in T Acute Lymphoblastic Leukemia (T-ALL) but the downstream elements that are able to support Notch-dependent leukemias are not well characterized. We have recently shown that the Notch-Hes1-CYLD-NFκB axis is crucial in the maintenance of T-ALL, but detailed evaluation of the contribution of each one of these elements is still missing. Here we use a Notch1-induced leukemia in vivo model to study the effect of silencing the Notch-target gene, Hes1 or overexpressing the Hes1-target, CYLD. We here show that both strategies completely abolish the ability of constitutive active Notch1 to generate T-ALL.

Introduction

The Notch pathway is responsible for the regulation of many cellular processes such as differentiation, proliferation, apoptosis and self-renewal. Activation of the pathway requires the association of one of the four different transmembrane Notch receptors (Notch1–4) with specific ligands present in neighboring cells. Following ligand binding, the Notch receptor undergoes two proteolytic cleavages, which result in the release of the intracellular domain of Notch (ICN). Then ICN is translocated to the nucleus where it forms a complex with RBPjκ and the coactivator mastermind (MAM) to activate the transcription of its target genes.¹ The principal targets of Notch are the members of the Hairy and Enhancer of Split (HES) family of transcriptional repressors.²

Activation of Notch has been associated with different types of human cancer such as intestinal carcinomas, gliomas, neuroblastomas^3,4 and leukemias.⁴ Specifically, activating mutations of Notch1 have been found in about 50% of human T-ALL.⁵ These mutations cluster in the heterodimerization (facilitating γ-secretase-dependent cleavage) or the PEST domain (increasing ICN1 protein stability).^5,6 Other mutations associated with T-ALL target the Fbw7 ubiquitin ligase that regulates Notch1 degradation⁷ further indicating the importance of Notch1 in these human leukemias. On the other hand, murine BM progenitors transduced with ICN1 generate T-ALL leukemias when transplanted into irradiated mice that are characterized by the presence of immature double positive (CD4⁺CD8⁺) T-cells in the peripheral blood and the bone marrow of the recipients.⁸

Despite the relevance of Notch1 activation in T-ALL pathogenesis, the molecular details of Notch-dependent oncogenesis are not completely understood. For example, it has been proposed that ICN1 can induce T-ALL through Hes1-induced repression of PTEN, which results in the activation of the PI3K/AKT survival pathway.⁹ On the other hand, it has been shown that Notch mutations in T-ALL are associated with activation of the NFκB pathway that is an important regulator of cell survival, proliferation and differentiation and it is frequently involved in malignant transformation.¹⁰ Activation of canonical NFκB pathway is produced through phosphorylation of the IκB inhibitors by the IKK complex of kinases. Phosphorylated IκB is then ubiquitinated and degraded via proteasome leading to the release of the NFκB complex that enters the nucleus to activate transcription.¹¹ Interestingly, IκBα is one the main transcriptional targets of NFκB activation, which results in a very effective negative feedback loop that prevents the pathway from chronic activation. However, other recently described mechanisms participate in NFκB inactivation including the elimination of the K63-linked ubiquitination of RIP, TRAF or IKKγ by the A20 and CYLD de-ubiquitinases (reviewed in ref. 12). Recently, we have shown that activation of the NFκB pathway by ICN1 in T-ALL^13,14 is in part mediated by the Hes1-dependent repression of CYLD, which results in sustained IKK activity.¹⁵ Moreover, we demonstrated that elimination of the regulatory IKKγ subunit in vivo, completely abrogates already established T-ALL in mice. However, whether Hes1 suppression or restoration of CYLD levels in vivo has any effect on Notch-induced leukemia remains to be determined.

Here we show in an in vivo model, that both silencing Hes1 and overexpression of the Hes1-target CYLD, completely abolish the ability of ICN1 to generate T-ALL when transduced into BM progenitors.

Results and Discussion

T-ALL is a complex disease that involves alterations in different pathways, and we previously identified the Notch1/Hes1/CYLD/NFkB axes as a crucial contributor to T-ALL maintenance.¹⁵ Sequence analysis of the regulatory regions of CYLD indicated the existance of two different 5′ regions that flank alternative exons 1a and 1b (P1 and P2) that contain three Hes binding consensus, which are shown in Figure 1A. By ChIP experiments, we demonstrated that Hes1 specifically binds to region P2 of the CYLD promoter in T-ALL cells.¹⁵ Now, we have cloned both promoter regions in a luciferase-reporter construct and performed reporter expression assays in NIH-3T3 cells. Our results indicate that both promoter regions are susceptible to be repressed by Hes1 in a dose-dependent manner, compared with the pGL3 vector reporter alone (Fig. 1B). Hes1 is the best-characterized Notch-target gene; however, other pathways are also able to modify the expression of hes1, implicating that Notch may not be the only pathway responsible for T-ALL. Previous work from our group demonstrated that NFκB activity is able to increase hes1 levels,¹⁶ which together with inhibition of CYLD by Hes1 may result in a feed-forward loop that might be crucial in leukemic maintenance. Moreover, we showed that inhibition of the NFκB pathway by genetic deletion of the IKKγ-element results in the abrogation of Notch1-induced leukemia.¹⁵ To specifically determine the contribution of Hes1 in the Notch-dependent leukemic transformation in vivo, we transduced bone marrow progenitors with ICN1 together with shRNA against hes1, plated them in methylcellulose and analyzed the cells after one week (Fig. 2A). As expected, constitutive activation of Notch1 increased the transcription levels of Hes1, concomitant with a decrease in CYLD expression in these cells. In contrast, knocking down hes1 with specific shRNA abolished the inhibition of CYLD by ICN1 (Fig. 2B). Importantly, ICN1 expressing cells were able to grow when serially replated in methylcellulose cultures, whereas elimination of hes1 by shRNA treatment significantly reduced this capacity (Fig. 2C and reviewed in ref. 15). To better understand the effect of Notch and Hes1 in this assay, we determined the percentage of cells that maintained the undifferentiated marker CD34 in each condition. We found that ICN1 significantly increased the amount of CD34⁺ cells compared with the controls and this percentage was reduced (P < 0.05) following hes1 knockdown (Fig. 2D). These results indicate that hes1 is an important mediator of the effects of Notch1 activation in self-renewal in vitro and suggest its involvement in maintaining leukemic stem cells. Next, we tested whether these cells were capable to generate leukemias in vivo when transplanted into lethally irradiated mice together with non-transduced bone marrow. In these experiments we found that ICN1-transduced cells generated T-cell leukemias characterized by the presence of circulating CD4⁺CD8⁺ double positive cells, whereas we did not detect any sign of leukemia neither in PB (Fig. 3A and B) nor in BM (Fig. 4B) in mice transplanted with ICN1 + shHES1 transduced cells. These results have further been confirmed in a different in vivo model by conditionally deleting hes1 in ICN1-induced leukemias.¹⁷ Altogether indicates that hes1 activation is absolutely required for the maintenance of Notch-dependent T-cell leukemias in vivo.

Figure 1. Hes1 represses CYLD promoter. (A) Scheme of the CYLD regulatory regions. (B) Luciferase assay with reporters of different CYLD promoters transfected with the Hes1 construct in NIH-3T3 cells. Graph is a representative from three experiments. Western blot analysis of Hes1 levels is shown in the lower part.

Figure 2. Hes1 is required for Notch-dependent T-cell transformation. (A) Experimental design: Lin-BM cells infected with the indicated viral constructs were serially replated during four weeks in methylcellulose media. RNA was extracted after week 1 and CD34 determination was performed after week 2. (B) Relative expression of the indicated genes in the different conditions. (C) Graph shows the cumulative number of colonies obtained after four weeks. (D) FACS analysis of cells expressing CD34 after week 2.

Figure 3. Hes1 is required for Notch-dependent T-cell leucemia in vivo. PB analysis after two months post-transplantation showing accumulation of double positive T lymphocyte from ICN1-transduced cells. (A) FACS analysis of CD4 and CD8 markers and (B) Giemsa staining of blood smears of a representative experiment.

Figure 4. CYLD expression abolishes Notch-dependent T-cell leukemias in vivo. PB analysis after two months post-transplantation of mice carrying cells transduced with the indicated constructs. (A) FACS analysis of CD4 and CD8 markers of a representative experiment. (B) Summary of the percentage of CD4⁺/CD8⁺ lymphocytes detected in BM of transplanted mice for each group from Figures 3 and 4.

Mutations in the Notch1 heterodimerization domain that give rise to ligand-independent but γ-secretase-dependent activation are present in about 50% of T-ALL patients. For this reason, pharmacological γ-secretase inhibitors (GSI) have become an obvious therapeutical opportunity for this type of leukemias. Consistent with this, several clinical trials are in progress (reviewed in ref. 18), but side effects due to drug toxicity are not yet solved, thus preventing the general use of these therapies. To avoid this problem different strategies are being tested to develop new and more specific inhibitors of the pathway.^19,20 On the other hand, despite ICN1 is sufficient to induce T-ALL in mice and the high correlation between the presence of activating Notch mutations and human T-ALL, a clear association between these mutation and patient prognosis or diagnosis has not been clearly demonstrated.²¹ This fact illustrates the possibility that leukemic T cells accomplish Notch activity through other strategies different from mutating the receptor. This is the case of human T-ALL carrying mutations in FBW7, the ubiquitin-ligase involved in ICN1 degradation.⁷ Another possibility is that Notch activity might not be required to maintain the tumor cells once the leukemia is established; however, our previous work showed that eliminating NFκB by specific hematopoietic deletion of IKKγ was sufficient to induce apoptosis in already established T-cell leukemias.

Recently, alterations in several NFκB members have been identified in multiple myeloma^22,23 and B-cell lymphomas, including the inactivation of the negative regulator of the pathway A20.^24,25 We have previously identified CYLD, an enzyme that is functionally equivalent to A20, as a downstream target of Hes1 repression in Notch-induced T-cell leukemias.¹⁵ We have now specifically tested the effect of CYLD re-expression in the capacity of ICN1 to induce T-cell leukemias in vivo. We transplanted lin-BM cells transduced with ICN1 alone or in combination with CYLD into irradiated mice and analyzed the presence of leukemic cells in PB and BM. Two months after transplantation, we found an accumulation of double CD4⁺/CD8⁺ cells in PB (Fig. 4A) and BM (Fig. 4B) of animals transplanted with ICN1 cells as expected. Importantly, this leukemic population was almost abolished by CYLD overexpression (P < 0.05) (Fig. 4A and B). These results demonstrate that CYLD inactivation, downstream of Hes1, displays a predominant role in maintaining T-ALL in vivo. Moreover, our data suggests that leukemic B-cells and T-cells preferentially exploit A20 or CYLD inactivation respectively, to maintain NFκB activity. In this sense, it has been shown that CYLD-deficiency specifically affects the normal differentiation of T-cells at the level of DP to SP transition.²⁶ Despite we have demonstrated that reverting CYLD is sufficient to prevent T-ALL in mice, our previous screening shows that both A20 and CYLD are downregulated in T-ALL human samples.¹⁵ These results indicate that A20 and CYLD might compensate each other in some situations or pathologies and require additional mechanisms to be inactivated. Thus, the mechanism involved in A20 inactivation in T-ALL should be a matter of future investigation, but in any case it is Hes1-independent.¹⁵

Altogether, our data indicate that NFκB is one of the most promising targets for cancer treatment since general activation of the pathway has been observed inducing multiple pro-oncogenic effects as well as chemotherapy resistance;^27,28 however, systemic side effects of NFκB inhibition have limited their application despite its clear involvement in this disease. In many tumors, NFκB is activated by cell extrinsic or microenvironmental factors, which complicates the specific implication of NFκB in the oncogenic phenotype. The identification of this new Notch-Hes1-CYLD-NFκB axes and its involvement in T-ALL opens the possibility of using a combination of NFκB and Notch inhibitors to treat this specific leukemia. In fact, glucocorticoids, which are widely used for T-ALL treatment are well-known, although non-specific, NFκB inhibitors²⁹ and it has been previously published that combination of GSI and glucocorticoids is effective on leukemic treatment in mice models while reducing the intestinal toxicity effects of Notch inhibition.³⁰ In addition, the proteasome inhibitor bortezomib, which is also a well-known NFkB inhibitor, is used for the treatment of multiple myeloma and mantle cell lymphoma.³¹

In summary, we here show that Hes1 directly represses CYLD promoter and that Hes1 activation and CYLD repression are both obligatory events to maintain T-ALL leukemic cells carrying active Notch in vivo. These results further support the development of NFκB-targeted therapies for specific oncologic diseases.

Materials and Methods

Mice

C57/BL6 mice were used and kept in pathogen-free animal facilities.

Plasmids

Mouse-specific Hes1-shRNA lentiviral constructs was purchased from Sigma (ref RCN0000028854). ICN1-GFP retroviral construct is a gift from I. Aifantis.¹⁴ CYLD lentiviral construct was given by A. Pfeifer.³²

Cytometry

For FACS experiments, the cells were collected in FBS 10% PBS, blocked in rat serum 20% PBS and stained 30 min at RT in FBS 10% PBS with BD PharMingen antibodies: PE-Cy7 Rat Anti-Mouse CD4, PE Rat Anti-Mouse CD8a, PE Rat Anti-Mouse CD34.

Erythrocytes were lysed with Pelilyse bufferA1 (Sanquin Plesmanlaan) before FACS analysis of PB.

Quantitative PCR

qRT-PCR was performed in LightCycler480 system using SYBR Green I Master or Probes Master kit (Roche). The primers used for RT-PCR analysis are listed in Table 1. GAPDH and ®2microglobulin are used as housekeeping.

Table 1. Primers used for RT-PCR analysis.

Gene	Primer forward	Primer reverse
Notch1	CAA TCA GGG CAC CTG TGA GCC CAC AT	TAG AGC GCT TGA TTG GGT GCT TGC GC
hes1	CGG CAT TCC AAG CTA GAG AAG G	Ggt agg tca tgg cgt tga tct g
CYLD	GCC TGG CTT TTC TTT GAC AG	GAA GGG CCA TCA TCA AAA GA
GAPDH	TGT TCC TAC CCC CAA TGT GT	TGT GAG GGA GAT GCT CAG TG
β2-microglobulin	CTG ACC GGC CTG TAT GCT AT	CAG TCT CAG TGG GGG TGA AT

Viral production

Recombinant lentivirus and retrovirus were produced by transient transfection of HEK-293T cells according to Tronolab protocols (tronolab.epfl.ch/page58122.html). Briefly, subconfluent HEK-293T were cotransfected with 20 μg of transfer vector, 15 μg of packaging plasmid (psPAX2) and 6 μg of envelope plasmid (pMD2.G). After 2 d, supernatant was ultracentrifuged in Beckman L-70 at 26,000 rpm for 2 h at 4 °C and viral pellet resuspended in 100 μl of PBS. Twenty μl of fresh viral suspension was used per infection.

Transduction of cells

BM cells from C57/BL6 mice were harvested and lineage depleted using the Biotin Mouse Linage Panel from BD PharMingen and anti-Biotin micro Beads from Miltenyi Biotec, according to manufacturing protocol. Lineage negative cells were cultured 3 d in Iscove’s 10% FBS, 1% Penicillin/streptomycin with the addition of Flt3L 50 ng/ml, SCF 50 ng/ml, IL6 10 ng/ml, IL7 10 ng/ml and transduced with ICN1-GFP retroviral construct or shRNA lentiviral constructs as previously described.³⁴ Cells were selected in methylcellulose with Hygromycin B (Invitrogen, 200 μg/ml) and Puromycin (Sigma, 0.5 μg/ml).

Transplantation assay

Cells were injected through the tail vein into lethally irradiated recipient as previously described in reference 33. Briefly, mice were irradiated with 9-Gy dose from a (137)Cs source and transplanted with 30,000–50,000 cells together with 500,000 non transduced BM cells. All animal procedures have been performed in accordance to the guidelines of the Institutional Animal Care and Use Committee of the PRBB and follow the regulation of Generalitat de Catalunya.