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Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Teo AE, Chen Z, Miranda RN, McDonnell T, Medeiros LJ, McCarty N - Leukemia (2015)

Bottom Line: On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6.Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells.Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation, Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas-Health Science Center at Houston, Houston, TX, USA.

ABSTRACT
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

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Decreased PAX5 levels in MCL cells promote lymphoma dispersal and can be used to predict a worse prognosis in MCL patients(A) CD19+ B cells were isolated from MCL biopsy or apharesis samples (n=31) or from normal blood (n=10), and the PAX5 mRNA levels were determined via qRT-PCR. Each replicate was performed in triplicate, and the expression levels were normalized to GAPDH. (B) CD19+ cells were purified from bone marrow of normal individuals (n=8) or MCL patients (n=8), and the PAX5 mRNA levels determined via qRT-PCR. Each replicate was performed in triplicate, and the expression levels were normalized to GAPDH. (C) PAX5 mRNA was downregulated in the bone marrow samples of MCL patients compared to the corresponding blood samples. (D) CD19+ cells isolated from GI-involved MCL patients (n=5) contained lower levels of PAX5 mRNA than CD19+ cells from non-GI-involved MCL patients (n=26). (E) CD19+ cells isolated from blastoid MCL patients (n=12) contained lower levels of PAX5 mRNA than CD19+ cells from non-blastoid MCL patients (n=19). A list of samples examined in (D) and (E) is presented in Supplementary Table 3. (F) The overall survival of MCL patients was significantly decreased in the PAX5low population compared to the PAX5high population (p < 0.05; Mantel-Cox curve analysis) (n=32). ‘PAX5high’ refers to the upper 50% of the PAX5 levels in the MCL patients and ‘PAX5low’ refers to who exhibit lower 50% of the PAX5 levels in the MCL patients. A list of samples is presented in Supplementary Table 5. *p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test). n.s. indicates a non-significant difference.
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Figure 5: Decreased PAX5 levels in MCL cells promote lymphoma dispersal and can be used to predict a worse prognosis in MCL patients(A) CD19+ B cells were isolated from MCL biopsy or apharesis samples (n=31) or from normal blood (n=10), and the PAX5 mRNA levels were determined via qRT-PCR. Each replicate was performed in triplicate, and the expression levels were normalized to GAPDH. (B) CD19+ cells were purified from bone marrow of normal individuals (n=8) or MCL patients (n=8), and the PAX5 mRNA levels determined via qRT-PCR. Each replicate was performed in triplicate, and the expression levels were normalized to GAPDH. (C) PAX5 mRNA was downregulated in the bone marrow samples of MCL patients compared to the corresponding blood samples. (D) CD19+ cells isolated from GI-involved MCL patients (n=5) contained lower levels of PAX5 mRNA than CD19+ cells from non-GI-involved MCL patients (n=26). (E) CD19+ cells isolated from blastoid MCL patients (n=12) contained lower levels of PAX5 mRNA than CD19+ cells from non-blastoid MCL patients (n=19). A list of samples examined in (D) and (E) is presented in Supplementary Table 3. (F) The overall survival of MCL patients was significantly decreased in the PAX5low population compared to the PAX5high population (p < 0.05; Mantel-Cox curve analysis) (n=32). ‘PAX5high’ refers to the upper 50% of the PAX5 levels in the MCL patients and ‘PAX5low’ refers to who exhibit lower 50% of the PAX5 levels in the MCL patients. A list of samples is presented in Supplementary Table 5. *p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test). n.s. indicates a non-significant difference.

Mentions: We conducted comprehensive PAX5 level analyses on 39 different primary MCL samples, consisting of 31 peripheral blood and 8 bone marrow aspirate specimens (Supplementary Table 3). All studies were conducted in a blinded manner, in which only CD19+ cells isolated via positive selection were analyzed. Compared to those from normal control blood or bone marrow samples, the PAX5 levels were significantly reduced in CD19+ cells from MCL patient samples (Figure 5a and b). Interestingly, the PAX5 levels in MCL CD19+ cells from bone marrow samples were significantly lower than those from blood samples (Figure 5c). To determine the experimental relevance of this result, we transplanted unmanipulated SP53 MCL cells into mice via subcutaneous injection. We observed that the PAX5 levels were significantly lower in disseminated human cells isolated from the spleen and bone marrow than in the localized parental subcutaneous tumor cells (Supplementary Figure 5a). PAX5− MCL xenografted mice displayed tumor spread to gastrointestinal sites (Supplementary Figure 3c). Similarly, we found that the PAX5 levels in CD19+ tumor cells were significantly lower in patients with a record of gastrointestinal involvement than in patients without recorded gastrointestinal involvement (Figure 5d).


Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Teo AE, Chen Z, Miranda RN, McDonnell T, Medeiros LJ, McCarty N - Leukemia (2015)

Decreased PAX5 levels in MCL cells promote lymphoma dispersal and can be used to predict a worse prognosis in MCL patients(A) CD19+ B cells were isolated from MCL biopsy or apharesis samples (n=31) or from normal blood (n=10), and the PAX5 mRNA levels were determined via qRT-PCR. Each replicate was performed in triplicate, and the expression levels were normalized to GAPDH. (B) CD19+ cells were purified from bone marrow of normal individuals (n=8) or MCL patients (n=8), and the PAX5 mRNA levels determined via qRT-PCR. Each replicate was performed in triplicate, and the expression levels were normalized to GAPDH. (C) PAX5 mRNA was downregulated in the bone marrow samples of MCL patients compared to the corresponding blood samples. (D) CD19+ cells isolated from GI-involved MCL patients (n=5) contained lower levels of PAX5 mRNA than CD19+ cells from non-GI-involved MCL patients (n=26). (E) CD19+ cells isolated from blastoid MCL patients (n=12) contained lower levels of PAX5 mRNA than CD19+ cells from non-blastoid MCL patients (n=19). A list of samples examined in (D) and (E) is presented in Supplementary Table 3. (F) The overall survival of MCL patients was significantly decreased in the PAX5low population compared to the PAX5high population (p < 0.05; Mantel-Cox curve analysis) (n=32). ‘PAX5high’ refers to the upper 50% of the PAX5 levels in the MCL patients and ‘PAX5low’ refers to who exhibit lower 50% of the PAX5 levels in the MCL patients. A list of samples is presented in Supplementary Table 5. *p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test). n.s. indicates a non-significant difference.
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Figure 5: Decreased PAX5 levels in MCL cells promote lymphoma dispersal and can be used to predict a worse prognosis in MCL patients(A) CD19+ B cells were isolated from MCL biopsy or apharesis samples (n=31) or from normal blood (n=10), and the PAX5 mRNA levels were determined via qRT-PCR. Each replicate was performed in triplicate, and the expression levels were normalized to GAPDH. (B) CD19+ cells were purified from bone marrow of normal individuals (n=8) or MCL patients (n=8), and the PAX5 mRNA levels determined via qRT-PCR. Each replicate was performed in triplicate, and the expression levels were normalized to GAPDH. (C) PAX5 mRNA was downregulated in the bone marrow samples of MCL patients compared to the corresponding blood samples. (D) CD19+ cells isolated from GI-involved MCL patients (n=5) contained lower levels of PAX5 mRNA than CD19+ cells from non-GI-involved MCL patients (n=26). (E) CD19+ cells isolated from blastoid MCL patients (n=12) contained lower levels of PAX5 mRNA than CD19+ cells from non-blastoid MCL patients (n=19). A list of samples examined in (D) and (E) is presented in Supplementary Table 3. (F) The overall survival of MCL patients was significantly decreased in the PAX5low population compared to the PAX5high population (p < 0.05; Mantel-Cox curve analysis) (n=32). ‘PAX5high’ refers to the upper 50% of the PAX5 levels in the MCL patients and ‘PAX5low’ refers to who exhibit lower 50% of the PAX5 levels in the MCL patients. A list of samples is presented in Supplementary Table 5. *p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test). n.s. indicates a non-significant difference.
Mentions: We conducted comprehensive PAX5 level analyses on 39 different primary MCL samples, consisting of 31 peripheral blood and 8 bone marrow aspirate specimens (Supplementary Table 3). All studies were conducted in a blinded manner, in which only CD19+ cells isolated via positive selection were analyzed. Compared to those from normal control blood or bone marrow samples, the PAX5 levels were significantly reduced in CD19+ cells from MCL patient samples (Figure 5a and b). Interestingly, the PAX5 levels in MCL CD19+ cells from bone marrow samples were significantly lower than those from blood samples (Figure 5c). To determine the experimental relevance of this result, we transplanted unmanipulated SP53 MCL cells into mice via subcutaneous injection. We observed that the PAX5 levels were significantly lower in disseminated human cells isolated from the spleen and bone marrow than in the localized parental subcutaneous tumor cells (Supplementary Figure 5a). PAX5− MCL xenografted mice displayed tumor spread to gastrointestinal sites (Supplementary Figure 3c). Similarly, we found that the PAX5 levels in CD19+ tumor cells were significantly lower in patients with a record of gastrointestinal involvement than in patients without recorded gastrointestinal involvement (Figure 5d).

Bottom Line: On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6.Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells.Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation, Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas-Health Science Center at Houston, Houston, TX, USA.

ABSTRACT
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

Show MeSH
Related in: MedlinePlus