Chronic lymphocytic leukemia (CLL) patients with deletion of chromosome 17p, where the p53 gene is located, often develop more aggressive disease with poor clinical outcomes. To investigate the underlying mechanisms for the highly malignant phenotype of 17p- CLL and to facilitate in vivo evaluation of potential drugs against CLL with p53 deletion, we have generated a mouse model with TCL1-Tg:p53−/− genotype. These mice develop B-cell leukemia at an early age with an early appearance of CD5+/IgM+ B cells in the peritoneal cavity and spleen, and exhibit an aggressive path of disease development and drug resistance phenotype similar to human CLL with 17p deletion. The TCL1-Tg:p53−/− leukemia cells exhibit higher survival capacity and are more drug resistant than the leukemia cells from TCL1-Tg:p53wt mice. Analysis of microRNA expression reveals that p53 deletion resulted in a decrease of miR-15a and miR-16-1, leading to an elevated expression of Mcl-1. Primary leukemia cells from CLL patients with 17p deletion also show a decrease in miR-15a/miR-16-1 and an increase in Mcl-1. Our study suggests that the p53/miR15a/16-1/Mcl-1 axis may be an important pathway that regulates Mcl-1 expression and contributes to drug resistance and aggressive phenotype in CLL cells with loss of p53.
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western countries, occurs with higher incidence in males, and is characterized by aberrant accumulation of neoplastic CD5+ B cells.1 Despite major progress in our understanding of CLL biology and the pathological processes, and the development of new drugs in the recent years, CLL remains an incurable disease.2 Cytogenetic alterations such as chromosomal aberrations are rather common in CLL and have been observed in the majority (∼80%) of CLL patients.3 Some of the cytogenetic changes are associated with poor prognosis and aggressive disease progression. Of note, chromosome 17p deletion (17p–) is a prominent cytogenic alteration in CLL associated with resistance to chemotherapy and poor clinical outcomes.4 As the tumor suppressor p53 gene is located in human chromosome 17p,5 it is suspected that the loss of p53 function in CLL cells with 17p– may be responsible for the poor prognosis of this subgroup of CLL patients.6, 7 Interestingly, recent study suggests a very high concordance (over 70%) in 17p deletion and mutations in the remaining p53 allele.8 Furthermore, p53 dysfunction may also arise via alternative mechanisms such as functional inactivation, which may explain certain CLL cases with poor prognosis but without apparent structural changes in p53 gene such as 17p deletion or mutations.9 Thus, it seems clear that the loss of p53 function has a profound effect on CLL response to drug treatment and the disease development. However, the underlying mechanisms remain to be elucidated.
Animal models are important tools to investigate disease processes and the associated pathological mechanisms in vivo. Currently there are several CLL mouse models, which include TCL1 transgenic mice,10 APRIL transgenic mice,11 Bcl-2 transgenic mice,12 the miR-155 mouse model,13 the NZB mouse model with miR-16 alteration14 and the miR-29 transgenic mice.15 The TCL1-Tg mouse model, which was created by the insertion of the human TCL1 gene under the control of the immunoglobulin heavy-chain variable region promoter and immunoglobulin heavy-chain enhancer, represents a commonly used and well-characterized mouse model that develops leukemia resembling human CLL.10 Previous studies have shown overexpression of B-cell lymphoma-2 (Bcl-2) family members in many cases of CLL, and this is correlated with resistance to therapy and a poor prognosis.16 In particular, the myeloid cell leukemia-1 (Mcl-1), one of the Bcl-2 family proteins, has been demonstrated as an important anti-apoptotic protein in CLL both in vitro and in vivo.17 It has been shown that Mcl-1 promotes CLL cell survival by inhibiting the intrinsic Bak/Bax-mediated apoptotic pathway.18 Loss of p53 function in cancer cells has also been associated with decrease in apoptotic response and drug resistance,19 and mice with p53−/− genotype are highly susceptible to the development of a variety of tumors.20 However, currently it is unclear if there is a link between the loss of p53 and overexpression of Mcl-1 in CLL cells.
In the present study, we generated a mouse colony with TCL1 transgenic and p53 deletion (TCL1-Tg:p53−/−) genotype by crossing the TCL1-Tg mice with p53−/− mice. The TCL1-Tg:p53−/− mice develop leukemia that seems to resemble human aggressive CLL disease with drug-resistant phenotype. The leukemia cells from TCL1-Tg:p53−/− mice exhibited higher proliferation, higher survival capacity, and more resistance to drug treatment with fludarabine (F-ara-A) than the leukemia cells from the TCL1 transgenic mice. We further demonstrated that the loss of p53 led to a significant increase in Mcl-1 expression, likely through the expression of miR-15a and miR-16-1. The association between the loss of p53, the decrease in miR-15a and miR-16-1 expression, and the increase in Mcl-1 was further confirmed in primary leukemia cells from CLL patients with chromosome 17p deletion. This study provides in vivo evidence to support a potential role of p53→miR15a/16-1→Mcl-1 axis in regulation of CLL cell survival and drug resistance, and this may contribute to the development of aggressive CLL.
Materials and Methods
9-β-D-arabinofuranosyl-2-fluoro-adenine (F-ara-A, the nucleoside form of fludarabine), oxaliplatin, chlorambucil, propidium iodide (PI) and PCR primers were purchased from Sigma-Aldrich (St Louis, MO, USA). Ficoll-lite Lympho H was from Atlanta Biological (Lawrenceville, GA, USA). CD19 microbeads were purchased from MACS Miltenyi Biotech Inc. (Auburn, CA, USA). ACK lysis buffer and Annexin-V-FITC were from BD Biosciences (San Jose, CA, USA). TUNEL staining kit was obtained from Roche Applied Science (Indianapolis, IN, USA). Antibodies against Bcl-XL, Bcl-2 and β-actin were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-Mcl1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Isolation of CLL cells and cytotoxicity assays
Primary leukemia cells (white blood cells) were isolated from the peripheral blood samples of CLL patients diagnosed according to the NCI criteria.21 Proper informed consent under a research protocol approved by the Institutional Review Board (IRB) of MD Anderson Cancer Center was obtained from all patients before the collection of blood samples. Specimens from CLL patients with or without 17p deletion were all used for comparison. CLL cells were isolated from blood samples by density gradient centrifugation as described previously,22 and incubated in RPMI 1640 medium supplemented with 10% FBS and penicillin (100 U/ml)+streptomycin (100 μg/ml) overnight before testing drug sensitivity by incubation with F-ara-A or oxaliplatin for 48 h. Peritoneal cavity (PC) cells and splenocytes were isolated and treated with ACK cell lysis buffer for 2 min on ice to remove red blood cells. After lysis, RPMI medium with 10% FBS was added to the cells to stop the lysis. Afterwards, the cells were washed once by PBS and filtered through a cell strainer with 40 μM nylon mesh (Fisher Scientific, Pittsburgh, PA, USA) for single-cell preparation, and cultured in the same medium as primary leukemia cells. B cells were purified from white blood cells by using CD19 microbeads, and incubated in RPMI 1640 medium supplemented with 10% FBS and penicillin (100 U/ml)+streptomycin (100 μg/ml). On the same day, those B cells were treated with F-ara-A or Oxaliplatin for 48 h. Cell viability and cellular sensitivity to drug treatment in vitro were determined by flow cytometry after double staining of 1 × 106 cells with annexin-V-FITC and PI as previously described.23
Mouse genotyping and analysis of cell-surface antigens
The generation of Eu-TCL1 mice and their maintenance were described previously.10 The Eu-TCL1-Tg homozygous mice (B6C3 strain) were mated with p53−/− mice (B6, 129S strain) to generate TCL1-Tg:p53+/− mice, which were further mated to generate mice with TCL1-Tg:p53−/− genotype. The first generated TCL1-Tg:p53−/− mice were further crossed to generate more TCL1-Tg:p53−/− mice for studies. All mice were housed in the conventional barrier animal facility at the University of Texas MD Anderson Cancer Center, and the animal study was carried out under a research protocol approved by the Institutional Animal Care and Use Committee (IACUC). For mouse genotyping, small segments of mouse tail tips were collected from littermates at the age of 3–4 weeks, and were digested in 200 μl tail lysis buffer (Viagen Biotech, Los Angeles, CA, USA) with 5 μl proteinase K at 56 °C in a water bath overnight, followed by a 5-min incubation at 95 °C and then cooled on ice. After removal of tissue debris by centrifugation, 2 μl supernatant was used in a PCR reaction for genotyping as described previously,10 and p53 genotyping protocol was provided by Chad Smith (Transgenic Core Facility of MD Anderson Cancer Center). Blood samples were collected from the mouse tails for white blood cell counting and analysis performed by the pathology service in the animal facility at MD Anderson Cancer Center. To analyze cell-surface CD5 and IgM, single-cell suspensions were prepared from the mouse spleen, bone marrow and the PC washout. The cells were stained for surface expression of CD5 and IgM using allophycocyanin (APC)-labeled anti-CD5 and FITC-labeled anti-IgM antibodies (Ebioscience, San Diego, CA, USA).
Analysis of cell proliferation and apoptosis
Mouse spleen sections were fixed in neutral buffered 10% formalin solution for preparation of tissue slides. Cell proliferation was estimated by Ki67 immunostaining using Ki67 specific antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody to reveal the diaminobenzidine (DAB) staining (Ki67 staining service ordered from the histology lab in MD Anderson Cancer Center, Houston, TX, USA). Terminal deoxynucleotidyl transferase deoxyuridine-triphosphatase nick-end labeling (TUNEL) assays were performed with an In Situ Cell Death Detection kit (Roche) according to the manufacturer’s instruction, and visualized under fluorescent microscopy. Annexin-V/propidium iodide (PI) double-staining and flow cytometry analysis were used to monitor cell death.
Analysis of microRNA expression
Total RNA was isolated from spleen cells from TCL1-Tg and TCL1-Tg:p53−/− mice or from human CLL cells with or without 17p deletion, using a microRNA isolation kit (Ambion, Grand Island, NY, USA). For analysis of microRNA expression profiles, 5 μg total RNA isolated from three mice or three CLL patient samples per group was analyzed using a commercial microRNA array (LC Sciences, Houston, TX, USA). For real-time PCR analysis, total RNA from 1 × 107 splenocytes was isolated from TCL1-Tg or TCL1-Tg:p53−/− mice (four mice with each genotype), purified using a RNeasy Mini kit (Qiagen, Germantown, MD, USA), and quantified using an Ultrospec 3300 pro UV/visible spectrophotometer (Biochrom Ltd., Cambridge, UK). First-strand cDNA was synthesized from 0.5 μg total RNA using a commercial kit (RevertAid First Strand cDNA Synthesis Kit-Fermentas; Thermo Scientific Inc., Waltham, MA, USA) according to the manufacturer’s instructions. Real-time PCR was performed using the 7900 GT sequence detection system (ABI PRISM; Ambion, Grand Island, NY, USA). Each PCR was performed in a 25-μl volume on a 96-well optical plate for 2 min at 50 °C, followed by 10 min at 95 °C, then followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min, and a final 10 min at 72 °C. To independently validate the individual microRNA expression pattern from the microRNA array result, total RNA was converted to cDNA synthesis using Taqman MicroRNA Reverse Transcription kit and Taqman RT primers (Applied Biosystems, LLC, Foster city, CA, USA). MicroRNA-specific real-time PCRs were performed using Taqman Universal PCR Master Mix and Taqman small assays according to the manufacturer’s recommended protocol. The relative expression of specific microRNAs was calculated by the delta (deltaCt) method.
Primary CLL cells were isolated from patient samples using Fico reagent as described above. Mouse splenocytes were purified by ACK lysis buffer. Cell number was determined by a Coulter Z2 particle count and size analyzer (Beckman Coulter, Inc., Fullerton, CA, USA). The mouse splenocytes/PC cells or primary CLL cells/B cells with the same amount were lysed in protein lysis buffer containing a cocktail of protease inhibitors. The nuclei and cell debris were removed by centrifugation at 4 °C (13 000 r.p.m. for 5 min), and the supernatants were collected as protein lysates. The protein lysates were then heated at 95 °C for 5–15 min, and separated by SDS-PAGE followed by western blot analyses with antibodies specific for Bcl-XL, Mcl-1, Bcl-2, EZH2 and b-Myb.
Student t tests were used for testing the statistical difference between two groups of samples. Mouse survival curves by Kaplan–Meier plots were generated by Graphpad Prism software (GraphPad, San Diego, CA, USA), and the statistical significance was analyzed by the log-rank (Mantel–Cox) test. A P value of less than 0.05 was considered statistically significant.
TCL1-Tg:p53−/− mice develop aggressive CLL with early disease onset and short lifespan
P53 is one of the most frequently mutated genes in cancers. In human B-CLL, loss of p53 function has been associated with accelerated disease progression, poor prognosis and resistance to antitumor agents. Currently there is no CLL mouse model with loss of p53 for investigating the pathological process of this aggressive CLL. To create such an animal model, we used the well-characterized TCL1 transgenic CLL mice to cross breed with p53−/− mice to generate progenies that harbor TCL1-Tg:p53−/− genotype. The TCL1-Tg:p53−/− mice developed CLL disease with early disease onset at the age of ∼3 months, and most of the mice (17 out of the 20 mice examined) developed severe splenomegaly by 4–5 months, while the spleens of TCL1-Tg mice with wt p53 or p53+/− mice appeared relatively normal in size (Figure 1a). The significant difference in the weights of the spleens from 4-month-old TCL1-Tg mice (n=5) and 4-month-old TCL1-Tg:p53−/− mice (n=5) is shown in Figure 1b (P<0.01).
Histological examination of the representative spleen sections of six 4-month-old TCL1-Tg mice showed normal tissue architecture, whereas the histological sections of the spleens from six 4-month-old TCL1-Tg:p53−/− mice showed that the lymphoid follicles were ill-defined (Figure 1c). The germinal centers of the TCL1-Tg:p53−/− spleens exhibited histological features reminiscent of the proliferation centers characteristic of CLL/small lymphocytic lymphoma because of the presence of large lymphocytes with abundant eosinophilic cytoplasm. The red pulps in between the lymphoid follicles contained lymphoid cells with more abundant cytoplasm, granulocytes and megakaryocytes compared with red pulps seen in TCL1-Tg mouse spleen (Figure 1c). Furthermore, blood smear revealed significant expansion of the white blood cells (WBC) in TCL1-Tg:p53−/− mice compared with TCL1-Tg mice (Figure 1d). Most of the TCL1-Tg:p53−/− mice died at the age of 3–5 months, while most of the TCL1-Tg mice survived more than 12 months (Figure 1e). This was consistent with the clinical observations that CLL patients with 17p deletion have significantly shorter overall survival compared with the CLL patients without 17p deletion.24
As TCL1-Tg mice show features of human CLL with an increase in CD5+/IgM+ B cells in the spleen, PC and bone marrow,10 we compared CD5+/IgM+ cells in the spleen, PC and bone marrow of the wild-type mice, p53−/− mice, TCL1-Tg mice, TCL1-Tg:p53+/− mice and TCL1-Tg:p53−/− mice. Flow cytometry analysis showed that at the age of 4 months, the wild-type mice showed 8% CD5+/IgM+ cells in the PC, the TCL1-Tg mice had 21% CD5+/IgM+ cells, and the TCL1-Tg:p53+/− and TCL1-Tg:p53−/− mice had 35% and 41% CD5+/IgM+ cells in PC, respectively (Figure 2a). By 5 months, the CD5+/IgM+ cells in PC of TCL1-Tg:p53+/− and TCL1-Tg:p53−/− mice increased to 58% and 79%, respectively (Figure 2b). In the spleens of 5-month-old mice, the CD5+/IgM+ cells were undetectable in wild-type mice, 7% in TCL1-Tg mice, 15% in TCL1-Tg:p53+/− mice and 20% in TCL1-Tg:p53−/− mice (Figure 2c). These data were consistent with the early onset of CLL in TCL1-Tg:p53−/− mice (Figure 1). Of note, in TCL1-Tg:p53−/− mice the CD19+ B cells were ∼90% in the peritoneal exudates and ∼70% in the spleen. Interestingly, in the control p53−/− mice without TCL1 transgene, the CD5+/IgM+ cells were undetectable in the spleens at 4 months, and about 10% of the PC cells were CD5+/IgM+ (Supplementary Figure S1). This was similar to the wild-type mice at 4 months (8% CD5+/IgM+ cells in the PC, Figure 2a). In contrast, the TCL1-Tg:p53−/− mice had substantially more CD5+/IgM+ cells (over 40%) in the PC (Figure 2a), suggesting that the earlier development of CLL in the TCL1-Tg:p53−/− mice was a combined effect of TCL1 activation and loss of p53, and not due to p53 deletion alone.
We also examined the bone marrow involvement in the mice, and found that there was no significant number of CD5+/IgM+ cells detected in the bone marrow of all genotypes at 3 months. At 7 months, CD5+/IgM+ cells were still undetectable in the bone marrow of the four TCL1-Tg mice examined. However, a substantial number of CD5+/IgM+ cells (20%) was detected in the bone marrow of 7-month-old TCL1-Tg:p53+/− mice (Supplementary Figure S2). The TCL1-Tg:p53−/− mice died before 7 months.
Loss of p53 in CLL cells promotes proliferation and cell survival
Recent study suggested that CLL cells in TCL1-Tg mice may undergo accelerated cell proliferation accompanied by elevated cell apoptosis, thereby displaying a low accumulation of CLL cells and slow disease progression.25 As p53 has a pivotal role in regulation of cell proliferation and apoptosis in response to various stimuli, we examined if a loss of p53 might affect CLL cell proliferation in TCL1-Tg mice. Immunostaining of spleen tissue slides with the proliferation marker Ki-67 revealed that Ki67-positive cells were significantly higher in the spleens of TCL1-Tg:p53−/− mice compared to that of TCL1-Tg mice (Figures 3a and c), suggesting an increase in proliferation of the splenocytes in TCL1-Tg:p53−/− mice.
We then used TUNEL assay to compare in vivo apoptosis of splenocytes in TCL1-Tg and TCL1-Tg:p53−/− mice. TUNEL staining of the spleen tissue sections showed that TCL1-Tg:p53−/− mice had significantly less apoptotic cells in the spleen compared to that in the spleen of TCL1-Tg mice (Figures 3b and d). Consistently, annexin-V/PI double-staining of the splenocytes revealed that the isolated splenocytes from TCL1-Tg:p53−/− mice were less apoptotic when cultured in vitro for 24–72 h compared with the splenocytes isolated from TCL1-Tg mice cultured under identical conditions (data not shown). Taken together, these data suggest that the loss of p53 in TCL1-Tg mice seems to promote cell proliferation and decrease apoptosis. This might account for the much higher accumulation of CLL cells and rapid disease progression in TCL1-Tg:p53−/− mice.
Leukemia cells from TCL1-Tg:p53−/− mice or from CLL patients with 17p deletion are resistant to chemotherapeutic drugs
The observation that the leukemia cells isolated from TCL1-Tg:p53−/− mice exhibited less spontaneous apoptosis (Figures 3b and d) prompted us to speculate that the leukemia cells with loss of p53 might be less sensitive to apoptotic induction, and thus might be more resistant to chemotherapeutic agents. To test this possibility, we isolated splenocytes from the wild-type control mice and from TCL1-Tg, TCL1-Tg:p53+/− and TCL1-Tg:p53−/− mice, and then treated the cells with several standard anti-CLL chemotherapeutic agents in culture.26 As shown in Figure 4a, the splenocytes from the control and TCL1-Tg mice exhibited similar sensitivity to F-ara-A (active form of fludarabine, 10 μM), chlorambucil (10 μM) and oxaliplatin (10 μM). The loss of one p53 allele (TCL1-Tg:p53+/−) caused a moderate decrease in drug sensitivity, whereas the loss of both p53 alleles (TCL1-Tg:p53−/−) led to a significant resistance to all three chemotherapeutic agents (Figure 4a).
To further confirm the correlation between the loss of p53 and drug resistance in primary CLL cells from patients, we compared the drug sensitivity of primary leukemia cells from CLL patients with or without 17p deletion. Flow cytometry analysis showed that CLL cells isolated from patients without 17p deletion were sensitive to F-ara-A (10 μM) and oxaliplatin (10 μM), which caused a loss of 30–50% cell viability during the 48-h drug incubation (Figure 4b). For instance, in patient no. 1 the control cell showed 78% viability, and drug treatment led to a substantial decrease of viable cells (37–41%). Similar results were observed in CLL cells from patient no. 2. In contrast, CLL cells with 17p deletion were highly resistant to F-ara-A and oxaliplatin (Figure 4b, patients no. 3 and no. 4). For instance, in CLL patient sample no. 4, the control sample without drug treatment showed 87% viable cells. After treatment with F-ara-A or oxaliplatin, the cell viability remained at 85–86%. Similar drug resistance was observed in patient sample no. 3. Drug resistance was also observed in other patient samples with 17p deletion. Figure 4c shows the quantitative data on drug-induced cell death in five CLL patient samples with 17p deletion in comparison with five CLL patient samples without 17p deletion. Furthermore, we also compared the drug sensitivity in purified B-CLL cells with or without 17p deletion, and observed similar patterns of drug response in vitro. As shown in Supplementary Figure S3, purified B-CLL cells with 17p deletion were highly resistant to F-ara-A and oxaliplatin. Together, the data from experiments with leukemia cells from mice and from CLL patient blood samples consistently suggest that a loss of p53 leads to drug resistance to standard chemotherapeutic agents.
Loss of p53 in CLL cells promotes Mcl-1 expression associated with downregulation of miR-15a and miR-16-1
To investigate the mechanisms that contribute to the drug-resistant phenotype in CLL cells lacking p53, we first compared the expression of the anti-apoptotic Bcl-2 family members, including Bcl-2, Mcl-1 and Bcl-XL, in CLL cells isolated from the spleens and PCs of the TCL1-Tg and TCL1-Tg:p53−/− mice. Western blot analysis showed that the expression of these anti-apoptotic molecules increased to various degrees in the p53-null cells (Figure 5a), with the elevation of Mcl-1 protein being the most prominent event, which was detected in leukemia cells isolated from spleen and PC of TCL1-Tg:p53−/− mice. Bcl-XL protein levels were also increased in both the splenocytes and PC cells from TCL1-Tg:p53−/− mice compared with TCL1-Tg mice. Interestingly, the increased Bcl-2 was observed in the PC cells but not in splenocytes (Figure 5a). Real-time reverse transcriptase PCR (RT–PCR) analysis showed a significant increase in mRNA expression of Mcl-1, Bcl-XL, and Bcl-2 in the splenocytes and PC cells from TCL1-Tg:p53−/− mice compared with those from TCL1-Tg mice (Figures 5b–d).
Importantly, the increase in Mcl-1, Bcl-XL and Bcl-2 protein expression was also observed in primary lymphocytes isolated from the peripheral blood of CLL patients with 17p deletion (Figure 5e). The increase in Mcl-1 was consistently observed in multiple patient samples, whereas the expression of Bcl-XL and Bcl-2 showed some heterogeneity. Real-time RT–PCR analysis showed that the mRNA expression of these three molecules was also increased in primary lymphocytes from CLL patients with 17p deletion (Figure 5f). To further confirm the increase of Mcl-1 expression in CLL cells with 17p deletion, we purified CD19+ CLL cells from four patients with 17p deletion and three patients without 17p deletion, and compared the expression of Mcl-1, Bcl-XL and Bcl-2 at protein and RNA levels. As shown in Figure 6, the protein levels of Mcl-1 and Bcl-XL were increased in CLL cells with 17p deletion, while Bcl-2 expression was high in all seven samples regardless of 17p status. Consistently, the expression of Mcl-1 mRNA was significantly increased in the 17p deletion samples. However, the increase of Bcl-XL and Bcl-2 mRNA was not statistically significant between the two groups. In addition, we analyzed the expression of Mcl-1, Bcl-XL, and Bcl-2 mRNA and proteins in purified CD19+ CLL cells from a total of 12 CLL patient samples (six with 17p deletion and six without deletion), and consistently observed the upregulation of Mcl-1 in CLL cells with 17p deletion, whereas the expression of Bcl-XL and Bcl-2 was heterogeneous among the patient samples (Supplementary Figure S4).
We then investigated the possible mechanism by which loss of p53 led to increased expression of the Bcl-2 family members. As the expression of Bcl-2 family members has been shown to be regulated by certain microRNAs,27 we speculated that the loss of p53 might cause a change in microRNA expression leading to overexpression of Mcl-1, Bcl-XL and Bcl-2. To test this possibility, we first isolated total RNA from the splenocytes of TCL1-Tg and TCL1-Tg:p53−/− mice, and examined the expression profiles of microRNAs using the LC Sciences microRNA array analysis. Among the >300 microRNAs detected by the microarray analysis, miR-15a and miR-16-1 consistently showed a marked decrease in the splenocytes of TCL1-Tg:p53−/− mice compared to that of the TCL1-Tg mice. This seemed consistent with the previous observation that miR-15a and miR-16-1 might have a role in regulating the expression of Bcl-2 family members.27 The decrease of miR-15a and miR-16-1 in cells from TCL1-Tg:p53−/− mice was further confirmed by real-time RT–PCR using the leukemia cells isolated from the spleens and PCs of TCL1-Tg and TCL1-Tg:p53−/− mice. As shown in Figure 7a, there was a significant decrease in the expression of miR-15a and miR-16-1 in both splenocytes and PC cells from mice without p53. Importantly, the decrease in miR-15a/16 expression was further confirmed in primary CLL cells isolated from five patients with 17p deletion (Figure 7b). Consistently, similar results were observed in B cells isolated from human WBC (Figure 8). These data together suggest that suppression of miR15a/miR16-1 expression may be an important mechanism by which the loss of p53 enhances the expression of Mcl-1, Bcl-2 and Bcl-XL, leading to increased cell viability and drug resistance.
Recent progress in investigation of CLL biology and the development of new therapies such as F-ara-A-based regimens has led to significant improvements of therapeutic outcomes. However, a subpopulation of CLL patients, particularly those with loss of p53 function due to chromosome 17p deletion or/and p53 mutations, are refractory to the current therapeutic regimens and have poor clinical outcomes.24 The p53 gene (TP53) is among the most commonly mutated genes in human cancers.28, 29, 30, 31 Loss of p53 function can be due to deletion or mutations of the gene that encodes for p53, epigenetic silencing and functional inactivation. In CLL, chromosome 17p deletion and p53 mutations are well-documented mechanisms that lead to loss of p53 function associated with poor prognosis.24, 32 The exact mechanisms by which loss of p53 may lead to drug resistance and aggressive disease process with poor clinical outcomes in CLL remain illusive. Based on the important role of p53 in cell cycle control and regulation of apoptosis,33 it is generally speculated that a loss of p53 function may result in an impairment of cycle–cycle checkpoints and compromised apoptotic response, leading to disease progression and drug resistance. However, there has no conclusive evidence in vivo to support this notion, perhaps in part due to the lack of proper CLL animal model with loss of p53.
In the present study, we generated a mouse colony with TCL1-Tg:p53−/− genotype, and demonstrated that these mice developed leukemia that seems to resemble aggressive human CLL. The TCL1-Tg:p53−/− mice exhibited signs of CLL disease around 3 months, with early appearance of CD5+/IgM+ cells in the PC and spleen. The p53−/− mice without TCL1-Tg did not show much accumulation of CD5+/IgM+ cells in the spleen and PC in the early months. Most TCL1-Tg:p53−/− mice showed highly abnormal accumulation of WBC in the blood and developed severe splenomegaly at 3–4 months, and died before 6 months. This is in contrast with the TCL1-Tg mice, which develop CLL approximately at the age of 1 year, and the disease progresses slowly.10 In the TCL1-Tg:p53−/− mice, we observed a significant increase in lymphoid cell proliferation in the spleen and a decrease in apoptosis. This may explain why these mice had severe accumulation of leukemia cells and enlargement of the spleen at early age. As p53 has a key role in enhancing the expression of apoptotic molecules such as Bax and PUMA,34 loss of p53 function would significantly compromise this apoptotic pathway, leading to resistance to chemotherapeutic agents. In fact, we observed that the leukemia cells isolated from TCL1-Tg:p53−/− mice or from CLL patients with 17p deletion were resistant to standard anti-CLL drugs such as F-ara-A and oxaliplatin. Furthermore, CD19 positive B cells purified from CLL patients with 17p deletion were also resistant to F-ara-A and oxaliplatin.
An important observation in this study was the significant upregulation of Mcl-1 expression in the leukemia cells lacking p53. This was seen both in the leukemia cells from the TCL1-Tg:p53−/− mice and in primary CLL cells from patients with 17p deletion (Figure 5). Mcl-1 is a key anti-apoptotic protein in the Bcl-2 family, and this molecule is known to be particularly important for the survival of CLL cells.17 Thus, upregulation of Mcl-1 may have a major role in apoptosis resistance in CLL cells lacking p53, and the moderate increase in Bcl-XL and Bcl-2 expression may also contribute to the increased viability of leukemia cells in TCL1-Tg:p53−/− mice. As the increase in Mcl-1 expression was observed at mRNA and protein levels, it is likely that loss of p53 may promote Mcl-1 expression mainly at the transcriptional level. This is consistent with the observation that p53 transcriptionally represses Mcl-1.35, 36, 37
Interestingly, the microRNA expression levels of miR15a and miR-16-1 were significantly decreased in CLL cells with loss of p53. This was observed both in the TCL1-Tg:p53−/− mouse model and in primary CLL cells isolated from patients with 17p deletion. As Mcl-1 appears to be a target of miR-15a and miR-16-1,38 the decrease in these microRNAs would release their suppression on Mcl-1 expression, and thus would lead to elevated MCL-1 protein level. Thus, it is possible that p53 might regulate Mcl-1 expression by modulating miR15a/16. It has been shown previously that microRNAs may be involved in CLL pathogenesis and prognosis due to their function as oncogenes or tumor suppressors.39 For example, miR-15a/miR-16-1 is located in chromosome 13q14.3, a region that is frequently mutated or deleted in CLL patients and may affect CLL cell survival and drug resistance.27 Intriguingly, p53 may regulate the expression of multiple microRNAs, many of which are closely involved in cell cycle regulation, proliferation and apoptosis.40 Our initial study using microRNA array to examine the effect of loss of p53 on microRNA expression in the TCL1-Tg:p53−/− mice had identified miR-15a and miR-16-1 being significantly downregulated, which was further validated by real-time RT–PCR. Clearly, miR-15a/16-1 expression is significantly lower in splenocytes and PC cells from TCL1-Tg:p53−/− mice than that of TCL1-Tg mice. Although p53 may directly repress MCL1 transcription, our study suggests that the p53→miR-15a/16-1→Mcl-1 axis may also be an important pathway that regulates the expression of the anti-apoptotic molecule and contributes to CLL cell viability and drug resistance. The important role of the p53→miR-15a/16-1→Mcl-1 axis in the development of drug-resistant CLL merits further investigation in clinical setting.
In summary, our study has generated TCL1-Tg:p53−/− mice, which develop aggressive CLL with an early disease onset, likely due to increase in proliferation of the leukemia cells and decrease in apoptosis. The CLL cells lacking p53 exhibited low sensitivity to standard anti-CLL drugs. Our study also provided in vivo evidence that loss of p53 led to upregulation of MCL-1 expression, likely through the downregulation of miR-15a and miR-16-1, and thus released their suppression on Mcl-1 expression, although the loss of p53 may also lead to increased MCL-1 protein. The TCL1-Tg:p53−/− mouse colony may serve as a valuable animal model to further investigate the in vivo drug resistance and the pathogenesis of aggressive CLL due to loss of p53 function. In addition, as these mice develop leukemia at early age and die within 6 months, this animal model may be useful in testing new drugs for their in vivo therapeutic activity against aggressive CLL with loss of p53 function. It should be noted, however, that the hallmark of human CLL with 17p deletion/p53 mutation is mainly manifested as drug resistance and poor treatment response, not so much as rapid disease progression, although aggressive disease course may be seen in some patients. This caveat should be noted when interpreting results from study using the TCL1-Tg:p53−/− mouse model.
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We thank BA Hayes and R LaPushin for their assistance in handling CLL samples. This work was supported in part by grants CA085563, CA100428 and CA016672 from the National Institutes of Health, grant RP110322 from the Cancer Prevention and Research Institute of Texas (CPRIT), and a grant from the CLL Global Research Foundation.
Contributions: JL designed and performed research, analyzed data and drafted the manuscript; GC, L F, HP, FW, WZ, MAO, WL performed research and analyzed data; HA performed histology/pathology analysis; CMC contributed TCL1 transgenic mice; MJK identified clinical specimens, designed research and interpreted data; PH directed the study design, data analysis/interpretation and drafted the manuscript.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Leukemia website
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Liu, J., Chen, G., Feng, L. et al. Loss of p53 and altered miR15-a/16-1→MCL-1 pathway in CLL: insights from TCL1-Tg:p53−/− mouse model and primary human leukemia cells. Leukemia 28, 118–128 (2014). https://doi.org/10.1038/leu.2013.125
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