ABSTRACT
Acute myeloid leukemia (AML) is a disease associated with epigenetic dysregulation. 11q23 translocations involving the H3K4 methyltransferase MLL1 (KMT2A) generate oncogenic fusion proteins with deregulated transcriptional potential. The polymerase-associated factor complex (PAFc) is an epigenetic co-activator complex that makes direct contact with MLL fusion proteins and is involved in AML, however, its functions are not well understood. Here, we explored the transcriptional targets regulated by the PAFc that facilitate leukemia by performing RNA-sequencing after conditional loss of the PAFc subunit Cdc73. We found Cdc73 promotes expression of an early hematopoietic progenitor gene program that prevents differentiation. Among the target genes, we confirmed the protein arginine methyltransferase Prmt5 is a direct target that is positively regulated by a transcriptional unit that includes the PAFc, MLL1, HOXA9 and STAT5 in leukemic cells. We observed reduced PRMT5-mediated H4R3me2s following excision of Cdc73 placing this histone modification downstream of the PAFc and revealing a novel mechanism between the PAFc and Prmt5. Knockdown or pharmacologic inhibition of Prmt5 causes a G1 arrest and reduced proliferation resulting in extended leukemic disease latency in vivo. Overall, we demonstrate the PAFc regulates Prmt5 to facilitate leukemic progression and is a potential therapeutic target for AMLs.
Subject(s)
Gene Expression Regulation, Leukemic , Histone-Lysine N-Methyltransferase/genetics , Leukemia, Myeloid, Acute/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Oncogene Proteins, Fusion/genetics , Protein-Arginine N-Methyltransferases/genetics , Animals , Cell Line, Tumor , Cell Proliferation/genetics , Disease Progression , Epigenesis, Genetic , Female , G1 Phase Cell Cycle Checkpoints/genetics , Gene Knockdown Techniques , Histones/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Transgenic , Myeloid Ecotropic Viral Integration Site 1 Protein/genetics , Myeloid Ecotropic Viral Integration Site 1 Protein/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/metabolism , Transcription Factors , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
PTPN11 encodes the Shp2 non-receptor protein-tyrosine phosphatase implicated in several signaling pathways. Activating mutations in Shp2 are commonly associated with juvenile myelomonocytic leukemia but are not as well defined in other neoplasms. Here we report that Shp2 mutations occur in human acute myeloid leukemia (AML) at a rate of 6.6% (6/91) in the ECOG E1900 data set. We examined the role of mutated Shp2 in leukemias harboring MLL translocations, which co-occur in human AML. The hyperactive Shp2E76K mutant, commonly observed in leukemia patients, significantly accelerated MLL-AF9-mediated leukemogenesis in vivo. Shp2E76K increased leukemic stem cell frequency and affords MLL-AF9 leukemic cells IL3 cytokine hypersensitivity. As Shp2 is reported to regulate anti-apoptotic genes, we investigated Bcl2, Bcl-xL and Mcl1 expression in MLL-AF9 leukemic cells with and without Shp2E76K. Although the Bcl2 family of genes was upregulated in Shp2E76K cells, Mcl1 showed the highest upregulation in MLL-AF9 cells in response to Shp2E76K. Indeed, expression of Mcl1 in MLL-AF9 cells phenocopies expression of Shp2E76K, suggesting Shp2 mutations cooperate through activation of anti-apoptotic genes. Finally, we show Shp2E76K mutations reduce sensitivity of AML cells to small-molecule-mediated Mcl1 inhibition, suggesting reduced efficacy of drugs targeting MCL1 in patients with hyperactive Shp2.
Subject(s)
Drug Resistance, Neoplasm/genetics , Interleukin-3/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Mutation/genetics , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Neoplastic Stem Cells/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Animals , Apoptosis , Apoptosis Regulatory Proteins , Blotting, Western , Cell Proliferation , Clinical Trials, Phase III as Topic , Female , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred C57BL , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Neoplastic Stem Cells/drug effects , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
Lactobacillus bulgaricus LT4(0448) is a lysogenic strain from which a temperate bacteriophage can be induced by mitomycin C or UV irradiation. Lactobacillus lactis CNRZ 326 is an indicator strain for the temperate phage 0448, but this strain lyses only in the presence of Ca ions. A resistant culture developed secondarily after phage lysis and grew normally in MRS broth but again lysed abruptly if Ca ions were added after two or three transfers. This behavior of the secondary culture and its subcultures is explained by a heterogeneous and fluctuating bacterial population, including clones identical to L. lactis 326, which were sensitive to 0448 and which formed rough colonies, as does the indicator. The proportion of these clones increased in the course of transfers in MRS, explaining lysis when Ca was added. The population also included clones which formed smooth colonies (S clones). SI clones, which could not be induced by mitomycin C, were the major type in the initial culture, although they were sensitive to temperate phage 0448. The SI population then decreased and was gradually replaced by SII clones, inducible by mitomycin C and resistant to 0448. These SII clones were lysogenized clones, 326(0448), whose stability was confirmed by growth in the presence of an antiphage serum. When L. bulgaricus LT4(0448) was treated with mitomycin C, several cured LT4 clones were obtained that were related to the clones of the indicator L. lactis 326; they formed rough colonies. They also became sensitive to lytic phages or temperate phages active against L. lactis 326 and insensitive to lytic phages which lysed L. bulgaricus LT4(0448). This suggests that phage 0448 can lead to a lysogenic conversion of host strain LT4.
