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PRPF8 defects cause missplicing in myeloid malignancies.

Kurtovic-Kozaric A, Przychodzen B, Singh J, Konarska MM, Clemente MJ, Otrock ZK, Nakashima M, Hsi ED, Yoshida K, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Ogawa S, Boultwood J, Makishima H, Maciejewski JP, Padgett RA - Leukemia (2014)

Bottom Line: Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis.Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects.In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland, OH, USA.

ABSTRACT
Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

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Cell proliferation and viability following PRPF8 knockdown(A) Proliferation of cells with reduced PRPF8 levels. Growth curve axis (number of viable cells in 106/ml) is shown on left y-axis and days on x-axis. In both types of cells, knockdown of PRPF8 leads to increased viable-cell counts. Error bars are calculated as one standard error. (B) Colony formation of K562 and CD34+ cells with PRPF8 knockdown showing increased colony counts. Cells, 104 K452 and 105 CD34+, were plated in semi-solid culture supplemented with cytokines and counted after 4 and 10 days, respectively. (C) Caspase 9 activity assay shows that there is no increase in apoptotic activity in PRPF8 knockdown cells. (D) Effect of treatment with the pre-mRNA splicing inhibitor meayamycin on patient cells with deletion 17p (two primary samples, Del17p AML #1 and #2) and mutant PRPF8 patient cells as well as normal bone marrow cells (NBM) and the KG1 cell line.
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Figure 3: Cell proliferation and viability following PRPF8 knockdown(A) Proliferation of cells with reduced PRPF8 levels. Growth curve axis (number of viable cells in 106/ml) is shown on left y-axis and days on x-axis. In both types of cells, knockdown of PRPF8 leads to increased viable-cell counts. Error bars are calculated as one standard error. (B) Colony formation of K562 and CD34+ cells with PRPF8 knockdown showing increased colony counts. Cells, 104 K452 and 105 CD34+, were plated in semi-solid culture supplemented with cytokines and counted after 4 and 10 days, respectively. (C) Caspase 9 activity assay shows that there is no increase in apoptotic activity in PRPF8 knockdown cells. (D) Effect of treatment with the pre-mRNA splicing inhibitor meayamycin on patient cells with deletion 17p (two primary samples, Del17p AML #1 and #2) and mutant PRPF8 patient cells as well as normal bone marrow cells (NBM) and the KG1 cell line.

Mentions: To evaluate the effect of reduced PRPF8 expression on cellular proliferation, we knocked down PRPF8 mRNA levels using two shRNA lentivirus constructs (Supplementary Figure 4). Decreased expression levels of PRPF8 were associated with increased cellular proliferation (Figure 3A) and increased clonogenicity (Figure 3B). Control and knockdown cells showed indistinguishable activity in a caspase 9 assay indicating no difference in apoptosis (Figure 3C). When PRPF8 mutant and deletion cells were treated with meayamycin, a potent pre-mRNA splicing inhibitor which targets the splicing factor 3b complex,15 growth of PRPF8 defective cells were more susceptible to inhibition than normal bone marrow cells (Figure 3D). The AML cell line KG1 that is monosomic for chromosome 17 is also more sensitive to meayamycin than normal bone marrow cells (Figure 3D).


PRPF8 defects cause missplicing in myeloid malignancies.

Kurtovic-Kozaric A, Przychodzen B, Singh J, Konarska MM, Clemente MJ, Otrock ZK, Nakashima M, Hsi ED, Yoshida K, Shiraishi Y, Chiba K, Tanaka H, Miyano S, Ogawa S, Boultwood J, Makishima H, Maciejewski JP, Padgett RA - Leukemia (2014)

Cell proliferation and viability following PRPF8 knockdown(A) Proliferation of cells with reduced PRPF8 levels. Growth curve axis (number of viable cells in 106/ml) is shown on left y-axis and days on x-axis. In both types of cells, knockdown of PRPF8 leads to increased viable-cell counts. Error bars are calculated as one standard error. (B) Colony formation of K562 and CD34+ cells with PRPF8 knockdown showing increased colony counts. Cells, 104 K452 and 105 CD34+, were plated in semi-solid culture supplemented with cytokines and counted after 4 and 10 days, respectively. (C) Caspase 9 activity assay shows that there is no increase in apoptotic activity in PRPF8 knockdown cells. (D) Effect of treatment with the pre-mRNA splicing inhibitor meayamycin on patient cells with deletion 17p (two primary samples, Del17p AML #1 and #2) and mutant PRPF8 patient cells as well as normal bone marrow cells (NBM) and the KG1 cell line.
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Related In: Results  -  Collection

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Figure 3: Cell proliferation and viability following PRPF8 knockdown(A) Proliferation of cells with reduced PRPF8 levels. Growth curve axis (number of viable cells in 106/ml) is shown on left y-axis and days on x-axis. In both types of cells, knockdown of PRPF8 leads to increased viable-cell counts. Error bars are calculated as one standard error. (B) Colony formation of K562 and CD34+ cells with PRPF8 knockdown showing increased colony counts. Cells, 104 K452 and 105 CD34+, were plated in semi-solid culture supplemented with cytokines and counted after 4 and 10 days, respectively. (C) Caspase 9 activity assay shows that there is no increase in apoptotic activity in PRPF8 knockdown cells. (D) Effect of treatment with the pre-mRNA splicing inhibitor meayamycin on patient cells with deletion 17p (two primary samples, Del17p AML #1 and #2) and mutant PRPF8 patient cells as well as normal bone marrow cells (NBM) and the KG1 cell line.
Mentions: To evaluate the effect of reduced PRPF8 expression on cellular proliferation, we knocked down PRPF8 mRNA levels using two shRNA lentivirus constructs (Supplementary Figure 4). Decreased expression levels of PRPF8 were associated with increased cellular proliferation (Figure 3A) and increased clonogenicity (Figure 3B). Control and knockdown cells showed indistinguishable activity in a caspase 9 assay indicating no difference in apoptosis (Figure 3C). When PRPF8 mutant and deletion cells were treated with meayamycin, a potent pre-mRNA splicing inhibitor which targets the splicing factor 3b complex,15 growth of PRPF8 defective cells were more susceptible to inhibition than normal bone marrow cells (Figure 3D). The AML cell line KG1 that is monosomic for chromosome 17 is also more sensitive to meayamycin than normal bone marrow cells (Figure 3D).

Bottom Line: Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis.Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects.In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland, OH, USA.

ABSTRACT
Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

Show MeSH
Related in: MedlinePlus