Homozygous deletions of p16/CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase (MTAP). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP-deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP-deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP-deleted cancers.

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Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation1,2. Here we show that mTORC1 regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. Through the use of integrative metabolomics in a mouse model3 and human biopsies4 of prostate cancer, we identified alterations in tumours impacting on the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation was validated in murine and human cancer specimens. AMD1 was upregulated in prostate cancer specimens with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus5 exhibited a predominant decrease in AMD1 immunoreactivity that was associated to a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

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Introduction miRs are a class of small, noncoding RNAs (18-23 nt) that control gene expression at the posttranscriptional level by repressing translation or by promoting degradation of the target mRNAs. 1-3 miRs play essential roles in many biologic processes, including development of the immune system and immune response. 4,5 Moreover, deregulated expression of specific miRs is associated with a wide variety of diseases, including both solid and hematopoietic malignancies. 6,7 Waldenström macroglobulinemia (WM) is a low-grade lymphoproliferative disorder characterized by the presence of an IgM monoclonal protein in the blood and monoclonal small lymphocytes and lymphoplasmacytoid cells in the BM. 8 In chronic lymphocytic leukemia (CLL), a combination of predominant resistance to apoptosis and continuing proliferation leads to progressive accumulation of phenotypically mature malignant lymphocytes. 9 Both diseases are incurable, low-grade, nonHodgkin B-cell lymphomas.Epigenetics, including DNA methylation, chromatin remodeling, and miR-mediated regulation of gene expression, has been implicated recently in the pathogenesis of B-cell malignancies. 10,11 We and others have demonstrated that primary WM and CLL cells present with increased expression of miR-155. 12,13 Furthermore, overexpression of miR-155 in B cells of transgenic mice leads to polyclonal pre-B cell proliferation, followed by lymphoblastic leukemia/high-grade lymphoma, indicating that miR-155 plays a crucial role in the pathogenesis of B-cell malignancies and is therefore a potential target for therapeutic intervention. However, reports on pharmacologic inhibition of miR-155 in mouse models of B-cell lymphoma have been lacking.Locked nucleic acid (LNA) is a conformational analog of RNA in which the ribofuranose ring in the sugar-phosphate backbone is locked in an RNA-like, C3Ј-endo conformation. 14 This results in high binding affinity between single-stranded, LNA-modified anti-miR oligonucleotides and their complementary miR targets. Several studies have reported on the inhibition of miR function using high-affinity 15-to 16-nt LNA-modified DNA phosphorothioate oligonucleotides targeting the 5Ј region of the mature miR. [15][16][17][18][19][20][21] Furthermore, a recent study described a method for antagonizing miR function using 8-mer LNA oligonucleotides complementary to the miR seed region, which were called "tiny LNAs." 22 In the present study, we assessed the efficacy of an 8-mer seed-targeting anti-miR-155 in inhibiting miR-155 function in low-grade non-Hodgkin B-cell lymphoma cells in vitro and in a mouse xenograft model of WM in vivo. Methods Cells and reagentsPrimary WM cells were collected from the BM of WM patients using CD19 ϩ microbead selection (Miltenyi Biotec) with more than 90% purity, as confirmed by flow cytometric analysis with an mAb against human CD19 (BD Biosciences). 13 Similarly, CD19 ϩ cells were isolated from the BM and peripheral blood of 3 healthy donors and used as controls. Approval for these studies was obtained from...

We have previously shown clinical activity of a mammalian target of rapamycin (mTOR) complex 1 inhibitor in Waldenstrom macroglobulinemia (WM). However, 50% of patients did not respond to therapy. We therefore examined mechanisms of activation of the phosphoinosi-

Waldenströ m macroglobulinemia (WM) cells present with increased expression of microRNA-206 (miRNA-206) and reduced expression of miRNA-9*. Predicted miRNA-206-and -9*-targeted genes include histone deacetylases (HDACs) and histone acetyl transferases (HATs), indicating that these miRNAs may play a role in regulating histone acetylation. We were able to demonstrate that primary WM cells are characterized by unbalanced expression of HDACs and HATs, responsible for decreased acetylated histone-H3 and -H4, and increased HDAC activity. We next examined whether miRNA-206 and -9* modulate the aberrant expression of HDAC and HATs in WM cells leading to increased transcriptional activity. We found that restoring miRNA-9* levels induced toxicity in WM cells, supported by down-modulation of HDAC4 and HDAC5 and up-regulation of acetyl-histone-H3 and -H4. These, together with inhibited HDAC activity, led to induction of apoptosis and autophagy in WM cells. To further confirm that miRNA-9*-dependent modulation of histone acetylation is responsible for induction of WM cytotoxicity, a novel class of HDAC inhibitor (LBH589) was used; we confirmed that inhibition of HDAC activity leads to toxicity in this disease. These findings confirm that histone-modifying genes and HDAC activity are deregulated in WM cells, partially driven by the aberrant expression of miRNA-206 and -9* in the tumor clone. IntroductionWaldenström macroglobulinemia (WM) is a B-cell low-grade lymphoma characterized by an arrest of B cells after somatic hypermutation and before isotype class switching. 1,2 Characterization of the cytogenetic and genetic abnormalities in WM has led to the identification of the long-arm deletion on chromosome 6 (6qϪ) in approximately 35% of the patients with this disease through the use of fluorescent in situ hybridization. 3 However, other cytogenetic and chromosomal abnormalities that are common in plasma cell dyscrasias, including multiple myeloma or in other low-grade B-cell malignancies, are not present in WM. Gene expression profiling in these patients has also been able to show that there are minimal genetic changes, with an expression profile similar to chronic lymphocytic leukemia myeloma. 4 Therefore, multilevel characterization of this disease at the epigenetic level is necessary to better identify molecular abnormalities that lead to tumor progression and survival in this disease.Epigenetic alterations include methylation, histone acetylation, and microRNA (miRNA) regulation. 5 Histone acetylation is commonly deregulated in many cancers. The balance of nucleosomal histone acetylation leads to the transcriptional regulation of many genes: hypoacetylation is associated with a condensed chromatin structure, leading to the repression of gene transcription, and acetylation is associated with a more open chromatin structure and activation of transcription. 6,7 This balance is maintained by a tight regulation of the level of histone deacetylase (HDAC) and histone acetyl transferases (HATs). In many malignancies, this balan...