Emerging molecular networks in Burkitt's lymphoma

Burkitt's lymphoma (BL), one of the most aggressive tumors affecting humans, characterized by the constitutive activation of the Myc oncogene together with the alteration of many other genetic and epigenetic factors. Among them, the INK4a/ARF locus has been well documented to play a central role in BL. Recently, we have discovered that simultaneous deregulation of both DNA methylation patterns and the ubiquitin‐dependent proteolysis system is required to completely inactive the INK4/ARF locus, opening new possibilities for treating Burkitt's lymphoma. In this review, we integrate our discovery with the general view of BL and propose a new comprehensive approach to analyze and manage this aggressive disease. J. Cell. Biochem. 114: 35–38, 2012. © 2012 Wiley Periodicals, Inc.     

Che‐1 is targeted by c‐Myc to sustain proliferation in pre‐B‐cell acute lymphoblastic leukemia

Despite progress in treating B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), disease recurrence remains the main cause of treatment failure. New strategies to improve therapeutic outcomes are needed, particularly in high‐risk relapsed patients. Che‐1/AATF (Che‐1) is an RNA polymerase II‐binding protein involved in proliferation and tumor survival, but its role in hematological malignancies has not been clarified. Here, we show that Che‐1 is overexpressed in pediatric BCP‐ALL during disease onset and at relapse, and that its depletion inhibits the proliferation of BCP‐ALL cells. Furthermore, we report that c‐Myc regulates Che‐1 expression by direct binding to its promoter and describe a strict correlation between Che‐1 expression and c‐Myc expression. RNA‐seq analyses upon Che‐1 or c‐Myc depletion reveal a strong overlap of the respective controlled pathways. Genomewide ChIP‐seq experiments suggest that Che‐1 acts as a downstream effector of c‐Myc. These results identify the pivotal role of Che‐1 in the control of BCP‐ALL proliferation and present the protein as a possible therapeutic target in children with relapsed BCP‐ALL.     

Elucidating the Targets of Antileukemic Agents: Molecular Mechanisms of Mercaptopurine Action

Mercaptopurine and thioguanine are anticancer and immunosuppresive agents that exert their primary cytotoxic effects via incorporation of deoxythioguanosine into DNA. Elucidation of molecular mechanisms underlying the sensitivity/resistance to thiopurine drugs is essential to further improving the therapy for leukemia. Inherited deficiency in thiopurine S-methyltransferase activity determines idiosyncratic reactions to thiopurines. Isolation and cloning of mutant alleles from humans with thiopurine S-methyltransferase deficiency has helped to develop PCR-based genotyping assays for this disorder. Incorporation of deoxythioguanosine into DNA substantially alters DNA–protein interactions at several steps of DNA replication. Importantly, it prevents RNA hydrolysis by RNase H in the DNA–RNA heteroduplex, and decreases the thermal stability of the DNA duplexes.     

AMPK promotes survival of c‐Myc‐positive melanoma cells by suppressing oxidative stress

Although c‐Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used the Nras^Q61KINK4a^?/? mouse melanoma model to show that c‐Myc is essential for tumor initiation, maintenance, and metastasis. c‐Myc‐expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c‐Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c‐Myc‐positive melanoma cells activated and became dependent on the metabolic energy sensor AMP‐activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c‐Myc‐expressing melanoma cells, while AMPK activation protected against cell death of c‐Myc‐depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA database analysis of early‐stage human melanoma samples revealed an inverse correlation between C‐MYC and patient survival, suggesting that C‐MYC expression levels could serve as a prognostic marker for early‐stage disease.     

Phosphoinositide 3-kinase/AKT/mTORC1/2 signaling determines sensitivity of Burkitt's lymphoma cells to BH3 mimetics

Burkitt's lymphoma (BL), driven by translocation and overexpression of the c-MYC gene, is an aggressive, highly proliferative lymphoma, and novel therapeutic strategies are required to overcome drug resistance following conventional treatments. The importance of the prosurvival BCL-2 family member BCL-X(L) in BL cell survival suggests that antagonistic BH3-mimetic compounds may have therapeutic potential. Here, we show that treatment of BL cell lines with ABT-737 induces caspase-3/7 activation and apoptosis with varying potency. Using selective inhibitors, we identify phosphoinositide 3-kinase (PI3K) as a proproliferative/survival pathway in BL cells and investigate the potential of combined pharmacologic inhibition of both the BCL-2 family and PI3K signaling pathway. PI3K/AKT inhibition and ABT-737 treatment induced synergistic caspase activation, augmented BL cell apoptosis, and rendered chemoresistant cells sensitive. Targeting mTORC1/2 with PP242 was also effective, either as a monotherapy or, more generally, in combination with ABT-737. The combined use of a dual specificity PI3K/mTOR inhibitor (PI 103) with ABT-737 proved highly efficacious. PI 103 treatment of BL cells was associated with an increase in BIM/MCL-1 expression ratios and loss of c-MYC expression. Furthermore, blocking c-MYC function using the inhibitor 10058-F4 also induced apoptosis synergistically with ABT-737, suggesting that maintenance of expression of BCL-2 family members and/or c-MYC by the PI3K/AKT/mTOR pathway could contribute to BL cell survival and resistance to ABT-737. The combined use of BH3 mimetics and selective mTORC1/2 inhibitors may therefore be a useful novel therapeutic approach for the treatment of B-cell malignancy, including chemoresistant lymphomas.     

NUDT15 polymorphism and NT5C2 and PRPS1 mutations influence thiopurine sensitivity in acute lymphoblastic leukaemia cells

In chemotherapy for childhood acute lymphoblastic leukaemia (ALL), maintenance therapy consisting of oral daily mercaptopurine and weekly methotrexate is important. NUDT15 variant genotype is reportedly highly associated with severe myelosuppression during maintenance therapy, particularly in Asian and Hispanic populations. It has also been demonstrated that acquired somatic mutations of the NT5C2 and PRPS1 genes, which are involved in thiopurine metabolism, are detectable in a portion of relapsed childhood ALL. To directly confirm the significance of the NUDT15 variant genotype and NT5C2 and PRPS1 mutations in thiopurine sensitivity of leukaemia cells in the intrinsic genes, we investigated 84 B-cell precursor-ALL (BCP-ALL) cell lines. Three and 14 cell lines had homozygous and heterozygous variant diplotypes of the NUDT15 gene, respectively, while 4 and 2 cell lines that were exclusively established from the samples at relapse had the NT5C2 and PRPS1 mutations, respectively. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with DNA-incorporated thioguanine levels after exposure to thioguanine at therapeutic concentration. Considering the continuous exposure during the maintenance therapy, we evaluated in vitro mercaptopurine sensitivity after 7-day exposure. Mercaptopurine concentrations lethal to 50% of the leukaemia cells were comparable to therapeutic serum concentration of mercaptopurine. Both NUDT15 variant genotype and NT5C2 and PRPS1 mutations were significantly associated with mercaptopurine sensitivity in 83 BCP-ALL and 23 T-ALL cell lines. The present study provides direct evidence to support the general principle showing that both inherited genotype and somatically acquired mutation are crucially implicated in the drug sensitivity of leukaemia cells.     

Myc-mediated proliferation and lymphomagenesis,but not apoptosis,are compromised by E2f1 loss

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Emu-Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either the Arf or p53 tumor suppressors. Emu-Myc transgenic mice lacking one or both E2f1 alleles exhibited a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27(Kip1) and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27(Kip1).     

The clinical impact of thiopurine methyltransferase polymorphisms on thiopurine treatment

Acute lymphoblastic leukaemia (ALL) is the most common malignancy of childhood. Although current treatment results in long term survival in over 70% of cases there is evidence that as many as 50% could have been cured using a less complex regimen with a lower incidence of long term side effects. In previous studies it has been found that thiopurines given as part of continuing therapy are key agents in preventing relapse. However, optimal administration during continuing therapy is often not achieved. Variation in the level of thiopurine methyltransferase (TPMT) activity appears to be a major molecular determinant of the extent of thiopurine metabolism. TPMT activity shows a trimodal distribution pattern. A lack of activity is found in approximately one in 300 Caucasians; approximately 11% have intermediate activity and the remaining 89% high activity. Congenital loss of activity is associated with grossly elevated levels of active drug and profound myelosuppression on exposure to thiopurines. This loss of activity has been attributed to single nucleotide polymorphisms (SNPs) within the TPMT gene. The frequency of SNPs is related to ethnicity, with the most common in Caucasians being TPMT*3A which is characterized by a G to A transition at position 460 with a substitution of alanine for tyrosine at amino acid 154 (A154Y) and a transition of A to G at nucleotide 719 resulting in a change of tyrosine to cysteine at position 240 (Y240C). Polymorphisms have also been identified within the 5' flanking promoter region of the TPMT gene due to a variable number of tandem repeats (VNTR*3-*8). An overview of the polymorphisms identified to date, their implication on the metabolism of the thiopurine drugs and therapeutic importance will be discussed.     

KSHV Latency Locus Cooperates with Myc to Drive Lymphoma in Mice

Kaposi sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi sarcoma and B-cell malignancies. Mechanisms of KSHV-induced oncogenesis remain elusive, however, in part due to lack of reliable in vivo models. Recently, we showed that transgenic mice expressing the KSHV latent genes, including all viral microRNAs, developed splenic B cell hyperplasia with 100% penetrance, but only a fraction converted to B cell lymphomas, suggesting that cooperative oncogenic events were missing. Myc was chosen as a possible candidate, because Myc is deregulated in many B cell lymphomas. We crossed KSHV latency locus transgenic (latency) mice to Cα Myc transgenic (Myc) mice. By itself these Myc transgenic mice develop lymphomas only rarely. In the double transgenic mice (Myc/latency) we observed plasmacytosis, severe extramedullary hematopoiesis in spleen and liver, and increased proliferation of splenocytes. Myc/latency mice developed frank lymphoma at a higher rate than single transgenic latency or Myc mice. These data indicate that the KSHV latency locus cooperates with the deregulated Myc pathways to further lymphoma progression.