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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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HTS of compounds reveals the drug-resistant nature of PAX5− MCL cells(A) Distribution of compounds examined via HTS that were cytotoxic to each cell type. The proliferation of control cells was inhibited by significantly more compounds than that of PAX5− cells. Compounds that displayed >40% cytotoxicity were classified as positive hits, and differences between the control and PAX5− cells (minimum of 5% cell cytotoxicity) were analyzed further. (B) A custom clinical library containing compounds with >10% (light gray) and >20% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. (C) Prestwick library of compounds with >10% (light gray) and >20% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. (D) NCI Diversity library featuring compounds with >20% (light gray) and >30% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. Only compounds that were more cytotoxic to PAX5− MCL cells were analyzed in (B), (C) and (D). (E) (Left) Jeko PAX5− cells were more resistant to compounds screened in the Custom Clinical Library (n=2). (Right) Inhibitors of the PI3K/mTOR and AKT/MEK/MAPK pathways less strongly induced the cytotoxicity of PAX5− MCL cells. The array on the right represents compounds targeting components of the aforementioned pathways in two different replicates.+ CONTROL = Compounds that were more sensitive towards control cells;+ PAX5− = Compounds more sensitive towards PAX5− cells;+ = compounds more resistant in PAX5− cells.
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Figure 6: HTS of compounds reveals the drug-resistant nature of PAX5− MCL cells(A) Distribution of compounds examined via HTS that were cytotoxic to each cell type. The proliferation of control cells was inhibited by significantly more compounds than that of PAX5− cells. Compounds that displayed >40% cytotoxicity were classified as positive hits, and differences between the control and PAX5− cells (minimum of 5% cell cytotoxicity) were analyzed further. (B) A custom clinical library containing compounds with >10% (light gray) and >20% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. (C) Prestwick library of compounds with >10% (light gray) and >20% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. (D) NCI Diversity library featuring compounds with >20% (light gray) and >30% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. Only compounds that were more cytotoxic to PAX5− MCL cells were analyzed in (B), (C) and (D). (E) (Left) Jeko PAX5− cells were more resistant to compounds screened in the Custom Clinical Library (n=2). (Right) Inhibitors of the PI3K/mTOR and AKT/MEK/MAPK pathways less strongly induced the cytotoxicity of PAX5− MCL cells. The array on the right represents compounds targeting components of the aforementioned pathways in two different replicates.+ CONTROL = Compounds that were more sensitive towards control cells;+ PAX5− = Compounds more sensitive towards PAX5− cells;+ = compounds more resistant in PAX5− cells.

Mentions: Compounds that displayed a difference in efficacy of at least 5% between control and PAX5− MCL cells were analyzed. After filtering the compounds displaying at least 40% of the inhibitory effect of doxorubicin (HTS positive control), we obtained a positive hit rate of 3.7% – 7.0% of the compounds in the HTS libraries. Across all three libraries, the PAX5− MCL cells were much more resistant than the control cells (Figure 6a). Further analysis revealed many unique compounds to which PAX5− MCL cells were resistant (Figure 6b–d); these compounds were selected based on their significance as detailed in the Supplementary Methods. We further analyzed the compounds to which PAX5− MCL cells were resistant and linked these compounds to known biological pathways. Compounds that targeted the PI3K–AKT-mTOR and MEK/ERK signaling cascades were less effective in killing PAX5− Jeko cells compared to control cells (Figure 6e). A similar trend was observed for PAX5− SP53 cells (Supplementary Figure 6e), suggesting that PAX5− MCL cells utilize hyperactivated PI3K–AKT-mTOR and MEK/ERK signaling pathways to promote their survival.


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)

HTS of compounds reveals the drug-resistant nature of PAX5− MCL cells(A) Distribution of compounds examined via HTS that were cytotoxic to each cell type. The proliferation of control cells was inhibited by significantly more compounds than that of PAX5− cells. Compounds that displayed >40% cytotoxicity were classified as positive hits, and differences between the control and PAX5− cells (minimum of 5% cell cytotoxicity) were analyzed further. (B) A custom clinical library containing compounds with >10% (light gray) and >20% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. (C) Prestwick library of compounds with >10% (light gray) and >20% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. (D) NCI Diversity library featuring compounds with >20% (light gray) and >30% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. Only compounds that were more cytotoxic to PAX5− MCL cells were analyzed in (B), (C) and (D). (E) (Left) Jeko PAX5− cells were more resistant to compounds screened in the Custom Clinical Library (n=2). (Right) Inhibitors of the PI3K/mTOR and AKT/MEK/MAPK pathways less strongly induced the cytotoxicity of PAX5− MCL cells. The array on the right represents compounds targeting components of the aforementioned pathways in two different replicates.+ CONTROL = Compounds that were more sensitive towards control cells;+ PAX5− = Compounds more sensitive towards PAX5− cells;+ = compounds more resistant in PAX5− cells.
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Related In: Results  -  Collection

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Figure 6: HTS of compounds reveals the drug-resistant nature of PAX5− MCL cells(A) Distribution of compounds examined via HTS that were cytotoxic to each cell type. The proliferation of control cells was inhibited by significantly more compounds than that of PAX5− cells. Compounds that displayed >40% cytotoxicity were classified as positive hits, and differences between the control and PAX5− cells (minimum of 5% cell cytotoxicity) were analyzed further. (B) A custom clinical library containing compounds with >10% (light gray) and >20% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. (C) Prestwick library of compounds with >10% (light gray) and >20% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. (D) NCI Diversity library featuring compounds with >20% (light gray) and >30% (dark gray) differences in cytotoxicity between the control and PAX5− MCL cells. Only compounds that were more cytotoxic to PAX5− MCL cells were analyzed in (B), (C) and (D). (E) (Left) Jeko PAX5− cells were more resistant to compounds screened in the Custom Clinical Library (n=2). (Right) Inhibitors of the PI3K/mTOR and AKT/MEK/MAPK pathways less strongly induced the cytotoxicity of PAX5− MCL cells. The array on the right represents compounds targeting components of the aforementioned pathways in two different replicates.+ CONTROL = Compounds that were more sensitive towards control cells;+ PAX5− = Compounds more sensitive towards PAX5− cells;+ = compounds more resistant in PAX5− cells.
Mentions: Compounds that displayed a difference in efficacy of at least 5% between control and PAX5− MCL cells were analyzed. After filtering the compounds displaying at least 40% of the inhibitory effect of doxorubicin (HTS positive control), we obtained a positive hit rate of 3.7% – 7.0% of the compounds in the HTS libraries. Across all three libraries, the PAX5− MCL cells were much more resistant than the control cells (Figure 6a). Further analysis revealed many unique compounds to which PAX5− MCL cells were resistant (Figure 6b–d); these compounds were selected based on their significance as detailed in the Supplementary Methods. We further analyzed the compounds to which PAX5− MCL cells were resistant and linked these compounds to known biological pathways. Compounds that targeted the PI3K–AKT-mTOR and MEK/ERK signaling cascades were less effective in killing PAX5− Jeko cells compared to control cells (Figure 6e). A similar trend was observed for PAX5− SP53 cells (Supplementary Figure 6e), suggesting that PAX5− MCL cells utilize hyperactivated PI3K–AKT-mTOR and MEK/ERK signaling pathways to promote their survival.

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