Insights about MYC and Apoptosis in B-Lymphomagenesis: An Update from Murine Models

The balance between cell survival and cell death represents an essential part of human tissue homeostasis, while altered apoptosis contributes to several pathologies and can affect the treatment efficacy. Impaired apoptosis is one of the main cancer hallmarks and some types of lymphomas harbor mutations that directly affect key regulators of cell death (such as BCL-2 family members). The development of novel techniques in the field of immunology and new animal models has greatly accelerated our understanding of oncogenic mechanisms in MYC-associated lymphomas. Mouse models are a powerful tool to reveal multiple genes implicated in the genesis of lymphoma and are extensively used to clarify the molecular mechanism of lymphoma, validating the gene function. Key features of MYC-induced apoptosis will be discussed here along with more recent studies on MYC direct and indirect interactors, including their cooperative action in lymphomagenesis. We review our current knowledge about the role of MYC-induced apoptosis in B-cell malignancies, discussing the transcriptional regulation network of MYC and regulatory feedback action of miRs during MYC-driven lymphomagenesis. More importantly, the finding of new modulators of apoptosis now enabling researchers to translate the discoveries that have been made in the laboratory into clinical practice to positively impact human health.

The MYC protein is organized as follow: a N-terminal transactivation domain (TAD), MYC box domains (MB0-IV), a PEST domain (Proline, glutamic acid (E), Serine and Threonine rich), a nuclear localization sequence (NLS), and the carboxy-terminus basic-helix-loop-helix-leucine zipper (bHLHZ). MYC protein domains participate in binding to a huge set of partner proteins, which modulate MYC functions and contribute to regulating gene expression. Specifically, MB0-II motifs affect the activity and stability of MYC and mediate the association of MYC with several proteins including P-TEFb, FBW7, and PIN1. MBIII-IV motifs of MYC are pivotal for the roles of MYC in apoptosis and in its protein turnover. Another MYC key domain is represented by the bHLHZ motif that is required for the interaction between MYC and MAX, essential for DNA binding [14].
The MYC gene is activated by growth factors and is sensitive to nutrient availability. In vitro studies confirmed that serum stimulation of quiescent cells in culture induced a rapid up-regulation of MYC mRNA. Conversely, MYC expression is reduced by nutrient deprivation and hypoxia [10,15]. The tight regulation of MYC expression in non-transformed cells was remarkably responsive to extracellular signals, at both transcriptional and translational levels [16]. Because MYC is one of the most highly regulated proteins in the cell, many important regulatory mechanisms have been discovered through studies of MYC [17].

MYC and Lymphomagenesis
MYC is one of the most deregulated oncogenes in humans and is frequently overexpressed in hematological malignancies due to aberrations. In most cases, aberrations include gene amplification or translocation, leading to MYC overexpression and a change in protein function or protein conformation [18,19]. MYC rearrangements have a high frequency in the aggressive B-cell lymphomas [20]. In Burkitt's lymphoma are present rearrangements in >90% of cases [21], in diffuse large B-cell lymphoma (DLBCL) about 5-14% of cases [22] and in Plasmablastic lymphoma (PBL) about 50% of cases [23]. MYC point mutations in the coding regions are another type of genetic aberration (e.g., in a majority of the Burkitt's lymphomas). They can induce MYC oncogenetic deregulation and can be resumed as follow: the residues most frequently mutated are Thr58 and S62 in the MBI motif [24]; the F138C mutation in the MBII motif [25]; and deletion of residues 188-199 in the MBIII motif [26].
A role for MYC in driving human cancer was first identified in Burkitt's lymphoma, where MYC deregulation was a result of chromosomal translocation into the immunoglobulin heavy chain locus [27]. MYC expression is deregulated and increased through different mechanisms in tumor cells. Several studies demonstrate unequivocally that the enhanced MYC expression is a common feature of tumorigenesis in both MYC-driven and non-MYC-driven tumors [28]. Recent studies suggest that lymphomagenesis in a pre-clinical mouse model Eµ-myc is also determined by MYC-cooperative mutations and concomitant multigenic lesions involving CDKN2a, NRAS, and KRAS, suggesting two-hit pathogenesis [29]. Combinations of MYC with other oncogenes including RAS [30], oncogenic EBV components [31], or MYC-surface CD19 signaling amplification loops have been shown to accelerate lymphoma development [32]. However, the suitability of these models to mirror therapeutic responses to target treatments has not been adequately tested. Notably, MYC promotes cell growth by stimulating the expression of the genes involved in the biogenesis of organelles such as ribosomes and mitochondria, glucose metabolism, and/or lipid synthesis. Furthermore, MYC is a pivotal player in the metabolic rewiring of cancer cells, the strategy that cancer cells adopt to cope with the energetic and metabolic demands required to support tumor growth and progression. [33][34][35]. MYC can contribute to tumorigenesis also influencing host immune cells [36]. Of note, MYC with STAT3 transcriptionally regulates PD-L1 in ALK-negative anaplastic large cell lymphoma (ALCL). In ALCL tumor-derived cell lines, MYC overexpression increased PD-L1 expression. Treatment with the BET inhibitor JQ1 or a MYC-targeting siRNA decreased PD-L1 expression [37]. In a panel of mouse MYC-driven lymphomas, tumor growth was dependent on MHC I expression that seemed to regulate innate immune surveillance [38,39].

MYC Expression as a Key Factor of Apoptosis Activation
MYC can promote cell activation, growth, and proliferation while concomitantly sensitizes cells to apoptosis. Normal cells have specific molecular sensors that enable detection of elevated MYC levels and response by triggering apoptosis. Conversely, transformed cells often acquire the ability to resist the apoptotic effects of elevated MYC and to respond, by increasing proliferation rates [17]. The abrogation of MYC-potentiated apoptosis is a crucial event of cellular transformation and cancer progression [40]. MYC can be conceived as a double face transcription factor since on one hand it supports cell proliferation and on the other apoptosis. Initially, it was considered disconcerting: a single molecule drives different functions. Quickly, these observations were confirmed for other oncogenes, including E2F1 and E1A [41]. Of note, premalignant cells allow an enhanced sensitivity to apoptosis caused by ectopic MYC expression, which they lose after malignant transformation [40]. Indeed, the tumor cells have acquired specific mechanisms for mitigating the apoptotic effects that MYC imposes in their normal counterparts. What follows are examples of key observations linking MYC to other apoptotic regulators.

Crosstalk between MYC and Cellular Regulators of Apoptosis in Lymphoma
It is well known that MYC contributes to oncogenic changes and cell transformation but its aberration alone maybe not sufficient to initiate lymphomagenesis. Important advances have been made in elucidating how cells respond to MYC activation by enhancing proliferation or by triggering apoptosis. These mechanisms are interconnected and involve two main pathways: BCL-2 pathway, p53 pathway, or both. In the beginning, apoptosis can be regulated by three distinct subgroups of proteins belong to the same family: BCL-2 proteins. They are clustered as follow: the pro-survival proteins (such as BCL-2, BCL-xL, MCL-1, and BCL-w) are essential for cell survival; the BH3-only proteins (for example, BIM and PUMA) are crucial for apoptosis initiation; finally, BAX/BAK are required for apoptosis execution [42]. MYC sensitizes pre-cancerous cells to undergo apoptosis by altering the balance of pro-and anti-apoptotic factors ( Figure 1). In the Eµ-myc model of lymphomagenesis, the Cleveland laboratory showed that MYC indirectly suppresses the anti-apoptotic proteins BCL2 and BCL-xL [43]. This is consistent with evidence showing that MYC triggers apoptosis through BAX. In this way, MYC activity directly influences cytochrome c release from the mitochondria, and therefore the activation of downstream effector caspases [42,44]. The BCL-2 gene is the first cell death regulator identified by its frequent translocation in a well-studied subtype of lymphoma in humans, the follicular B-cell lymphoma (FL) [45]. Its oncogenic potential was revealed when BCL-2 overexpression was shown to promote the survival (but not proliferation) of pro-B-cells deprived of cytokines [46]. DLBCLs contain both rearrangements in BCL-2 and translocation of MYC. They are classified as "double-hit lymphomas" (DHLs) and represent~10% of DLBCL cases [47]. Most recent discoveries have focused on the impact of two anti-apoptotic proteins of BCL-2 family, BCL-w and MCL-1 on MYC-driven lymphomagenesis. The overexpression of BCL-w was observed in DLBCL and correlated with poorer clinical outcomes. Of note, MYC can indirectly suppress BCL-w expression during lymphomagenesis, by up-regulating the miR-15 family members. Its loss profoundly delayed MYC-mediated B-cell lymphoma development due to increased B-cell apoptosis mediated by MYC. Loss of BCL-w sensitized B-cell to growth factor deprivation-induced B-cell apoptosis and suppressed MYC-induced lymphomagenesis, suggesting a crucial role of BCL-w in B-cell survival and lymphoma development [48]. MCL-1 protein attends in promoting the survival of B lymphoid progenitors that undergo MYC-driven lymphomagenesis. Loss of one MCL-1 allele almost abrogated MYC-driven-lymphoma development owing to a reduction in lymphoma initiating pre-B-cells. [49]. Moreover, MCL-1 is critical also for the development of B-cells lymphoma and its down-expression in lymphomas can be compensated by altered levels of other cell death regulators, such as BIM and/or BCL-xL [50]. enhancer elements that contribute to MYC deregulation. By transgenic and knock-in animal models that bring the oncogene myc under Eμ/3′RR transcriptional control, it was shown the essential contribution of both Eμ and 3′RR in B-cell lymphomagenesis. Of note, transgenic mice had a high rate of lymphoma emergence, increased proliferation, and the highest transcriptomic similarities to human BL [78]. Sabò et al. analyzed the genomic distribution of MYC during B-cell lymphomagenesis in the Eμmyc transgenic mice and generated ChIP-seq profiles. Among other genes, they observed that MYC associated with regulatory elements of the Ibtk gene. Intriguingly, MYC binding intensity of Ibtk promoter increased during lymphoma progression [79]. Ibtk is a gene coding for a protein belonging to the Cul3-dependent ubiquitin ligase (CRL3 IBTK ) complex [80,81] and, it has been shown to act mainly by counteracting B-cells apoptosis during B-tumorigenesis [82], especially during MYCdriven lymphomagenesis [83]. Of note, loss of Ibtk delays the tumor onset and improves animal survival, by reducing the number of pre-cancerous B-cells of bone marrow and spleen. It is due principally to impaired viability and increased apoptosis of pre-cancerous B-cells. Consequently, it was observed the altered expression of proteins leading to apoptotic pathways dependent on MYC overexpression in pre-cancerous Eμ-myc mice, such as p53 and MCL-1 protein. Furthermore, a recent report on Ibtk shows that its haploinsufficiency also alters tumor microenvironment by enhancing MYC-driven vascularization in B-cell lymphomas [33].
Recent papers show that lncRNAs are pivotal in MYC-driven B-lymphoproliferative disorders. An example is lncRNA NEAT1 (Nuclear Paraspeckle Assembly Transcript 1). The aberrant expression of lncRNA NEAT1 is a common feature of DLBCL [84] and chronic myeloid leukemia (CML) [85]. In the latter, it was shown that MYC can bind and suppress NEAT1 expression, On the other hand, p53 is one of the key molecules involved in the pathogenesis of B-cell lymphomas. Lymphomas with co-existent MYC and p53 alterations/mutations are synergistic, resulting in more aggressive lymphomas, and patients have a particularly poor prognosis with short median survival time [51]. Previous studies demonstrated that the deregulation of MYC up-regulates ARF, which in turn activates p53 to regulate a pool of target genes involved in apoptosis and growth arrest [52]. ARF-MDM2-p53 pathway in MYC-induced apoptosis accelerated tumorigenesis through the loss of these tumor suppressors in mouse models of MYC oncogenesis [53][54][55][56][57][58][59]. Notably, BMI-1 has been one of the first genes initially identified as an oncogene that cooperates with MYC in the generation of mouse pre-B-cell lymphomas by inhibiting INK4a/ARF proteins and by activating the human telomerase reverse transcriptase (h-TERT) [60].
One of the main p53 target genes is PUMA, a BH3-only pro-apoptotic protein. Previous studies observed that puma deletion in the Eµ-myc mouse accelerates MYC-driven lymphomagenesis [61]. Normally, PUMA initiates programmed cell death upon induction, triggering the cytochrome c release and caspase activation [62]. Conversely, deletion of puma efficaciously preserves cells against apoptosis induced by either DNA damage or oncogene activation, competing with the protection if p53 is deleted [61].

Transcriptional Regulation Network Involved in MYC-Driven Lymphomagenesis
Many other genes are involved in the pathogenesis of lymphoma cross-talking with MYC, mainly through transcriptional regulation (see in Table 1 and Figure 1). The first dimerization partner of MYC [63], MAX, have a role in MYC-induced oncogenesis. It is well to remember that overexpression of MAX alone in murine lymphoid cells is non-oncogenic and results in reduced B-cell proliferation. Noteworthy, B-cell-specific deletion of MAX has a modest effect on B-cell development but completely abrogates Eµ-myc-driven lymphomagenesis. Moreover, a study found that MAX loss leads to a significant reduction in MYC protein levels and down-regulation of direct transcriptional targets in premalignant Eµ-myc cells, including regulators of MYC stability [64]. Furthermore, the transcriptional repressor and MAX interactor MNT (MAX Network Transcriptional Repressor), competes with MYC for occupancy at E-box sequences in promoter regions [65]. Campbell and colleagues showed that MNT heterozygosity slowed MYC-driven tumorigenesis in vavP-MYC10 and Eµ-myc mice, suggesting that MNT facilitates MYC-driven oncogenesis, although no differences in the number of preleukaemic cells were apparent [66]. Recently, Nguyen and colleagues clearer explained the phenomenon. They showed that MNT supports B-lymphomagenesis by arresting MYC-induced apoptosis, primarily through suppressing BIM. In Eµ-myc mice, homozygous mnt deletion decreased lymphoma incidence by enhancing apoptosis and reducing premalignant B-cells [67]. Other transcriptional regulators have been described to antagonize or cooperate with proto-oncoprotein MYC. For example, a tumor suppressor PRDM11 has been shown to interact in vivo with MYC during lymphomagenesis. Loss of Prdm11 accelerates MYC-driven lymphomagenesis, regulating target genes in DLBCL cells such as FOS and JUN. Furthermore, patients with PRDM11-deficient DLBCLs have poorer survival [68]. A well-known transcription factor family, FOXO (Forkhead Box O) can also promote lymphomagenesis. Taking advantage of vavP-MYC10 and Eµ-myc mice, loss of FoxO3 variant induced significant acceleration of tumorigenesis. Loss of FoxO3 increased myeloid cells on the blood, spleen and, bone marrow of Eµ-myc mice, as in MYC10 mice. In vitro assays demonstrated that loss of FoxO3 had no impact on the cycling of preneoplastic and neoplastic Eµ-myc pre-B-cells, as well as, the selective pressure for mutation of the p53 pathway during lymphomagenesis was not altered [69]. Recent studies focused on FOXO1 protein [70], the highest FOXO protein expressed in lymphoid cells [71]. It was shown the abundant nuclear localization of FOXO1 in Burkitt's lymphoma (BL). Using genome editing in human and mouse MYC-driven lymphoma, the study demonstrates its pro-proliferative and anti-apoptotic activity in BL and identify its nuclear localization as an oncogenic event in germinal center B-cell derived lymphomagenesis [70]. In B-cell lymphomas, MYC gene can be inserted in various positions of the IgH locus. Once translocated and having lost its normal control, MYC is constitutively expressed throughout the cell cycle in B-cells. Two major IgH transcriptional enhancers have been reported so far to be linked with the oncogene MYC as the cause of B-cell malignancies. The Eµ enhancer controls early events in B-cell maturation such as VDJ recombination [72,73] while the 3 regulatory region (3 RR) controls late events in B-cell maturation such as IgH transcription [74], somatic hypermutation [75], and class switch recombination [76,77]. More recent reports have demonstrated that there are IgH intrachromosomal interactions between these 2 potent cis-acting enhancer elements that contribute to MYC deregulation. By transgenic and knock-in animal models that bring the oncogene myc under Eµ/3 RR transcriptional control, it was shown the essential contribution of both Eµ and 3 RR in B-cell lymphomagenesis. Of note, transgenic mice had a high rate of lymphoma emergence, increased proliferation, and the highest transcriptomic similarities to human BL [78].
Sabò et al. analyzed the genomic distribution of MYC during B-cell lymphomagenesis in the Eµ-myc transgenic mice and generated ChIP-seq profiles. Among other genes, they observed that MYC associated with regulatory elements of the Ibtk gene. Intriguingly, MYC binding intensity of Ibtk promoter increased during lymphoma progression [79]. Ibtk is a gene coding for a protein belonging to the Cul3-dependent ubiquitin ligase (CRL3 IBTK ) complex [80,81] and, it has been shown to act mainly by counteracting B-cells apoptosis during B-tumorigenesis [82], especially during MYC-driven lymphomagenesis [83]. Of note, loss of Ibtk delays the tumor onset and improves animal survival, by reducing the number of pre-cancerous B-cells of bone marrow and spleen. It is due principally to impaired viability and increased apoptosis of pre-cancerous B-cells. Consequently, it was observed the altered expression of proteins leading to apoptotic pathways dependent on MYC overexpression in pre-cancerous Eµ-myc mice, such as p53 and MCL-1 protein. Furthermore, a recent report on Ibtk shows that its haploinsufficiency also alters tumor microenvironment by enhancing MYC-driven vascularization in B-cell lymphomas [33]. Recent papers show that lncRNAs are pivotal in MYC-driven B-lymphoproliferative disorders. An example is lncRNA NEAT1 (Nuclear Paraspeckle Assembly Transcript 1). The aberrant expression of lncRNA NEAT1 is a common feature of DLBCL [84] and chronic myeloid leukemia (CML) [85]. In the latter, it was shown that MYC can bind and suppress NEAT1 expression, enhancing the imatinib-induced apoptosis [85]. MYC regulates cell proliferation of DLBCL via the NEAT1-miR-34b-5p-GLI1 signaling axis modulating DLBCL progression. A direct bound to the NEAT1 promoter allows MYC to regulate the NEAT1 level. In particular, knockdown of NEAT1 constrained cell proliferation and facilitated apoptosis of DLBCL cells through the miR-34b-5p-GLI1 pathway [86].

Regulatory Feedback Action of miRs during MYC-Driven Lymphomagenesis
Additionally, key microRNAs (miRs) (such as miR-34a and miR17-92) are related to MYC activation and play a vital role in some B-cell lymphomas [51]. The relevance of MYC-miRs circuits in B-cell functions is a novel insight into lymphomagenesis. MiRs interact with MYC acting as regulatory feedback that can influence the pre-or post-transcriptional expression of multiple genes, as shown in Table 2 and Figure 1. MiR-34a is reported to have tumor-suppressive activity in the oncogenesis of lymphoma and progression, contributing to p53-dependent apoptosis [87,88]. In DLBCL cells MYC mediated the repression of miR-34a resulting in a high proliferation of B-cell lymphoma by dysregulation of its target FOXP1 [89]. miR-34a and let7a are also targets of MYC repression establishing a feedback loop that aggravates and perpetuates the effects of MYC overexpression and may thus contribute to cancer progression [90]. MYC can indirectly repress several miRs by the recruitment of histone deacetylase (HDAC). It is known that the expression of miR-15a/miR-16-1 promotes apoptosis of cancer cells and suppresses tumorigenicity. The miR-15a/16-1 cluster directly down-regulates the anti-apoptotic protein BCL-2, MCL-1, CCND1, and WNT3A [91] and leads to induction of G0G1 arrest in tumor cells by suppressing cell cycle-positive regulators including CDK6 (Cyclin Dependent Kinase 6), Cyclins D and E [92]. Through epigenetic regulation and recruiting histone deacetylase 3 (HDAC3), MYC represses miR-15a/miR-16-1 expression in mantle cell and other non-Hodgkin B-cell lymphomas [93]. Indeed, MYC and EZH2 act in concert to silence tumor suppressor miRs in aggressive lymphoma cells. Of note, MYC recruited EZH2 to miR-26a promoter repressing miR-26a expression in aggressive lymphoma cell lines as well as primary lymphoma cells [94]. Concomitantly inhibitory action of both EZH2 and HDAC3 promoted transcriptional and epigenetic MYC-mediated repression of miR-29, resulting in down-regulation of miR-29 target pro-apoptotic genes and lymphoma growth suppression in vitro and in vivo [95]. MYC can work not only indirectly on the regulation of miRs but also as direct activator. The best example is the case of the oncogenic miR17-92 cluster. The miR17-92 polycistron at 13q31.3 is commonly amplified in several subtypes of aggressive lymphomas, including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), BL [96,97]. Several oncogenic transcription factors (TFs) regulate the expression of miR-17-92 cluster thus influencing its oncogenic activity. MYC was the first identified transcription regulator of miR-17-92 by directly binding to its genomic locus. The first model of MYC-miR-17-92 interaction was demonstrated in Eµ-miR-17~92 transgenic mice, where the overexpression of miR-17-19b promotes the oncogenesis of MYC expressing B-cell [98]. MYC may suppress the expression of the pro-apoptotic BIM gene, a target of miR-17-92 thus explaining its tumor-promoting function, so contributing to maintaining survival and self-renewal [5]. Furthermore, the induction of the miR-17-92 cluster by MYC attenuates E2F1 protein expression, such that interruption of this regulatory loop results in DNA replication stress thus promoting lymphomagenesis [99]. The high miR-17-92 expression has been found in BL, confirming that the activation of the MYC/miR-17-92 axis is a general feature of this disease [100]. Of note, the typical translocation of MYC into the immunoglobulin heavy chain locus is observed in about 80% of BLs [101] and this overexpression can induce higher miR-17-92 levels. Further studies are needed to investigate the role of miR-17-92 in DLBCL, where the pro-apoptotic PTEN tumor suppressor gene could be involved as miR-19a target [102]. As it is also a direct MYC target, MYC-induced miR may coordinate the balance of cell proliferation and cell death in these lymphomas. On the other hand, previous studies have suggested PTEN as an indirect regulator of MYC activity through activation of the PI3K-AKT-GSK3β pathway. Down-regulation of the PTEN protein may represent yet a pathway of post-transcriptional MYC activation [103].
Furthermore, MYC acts as a negative regulator of miR-144/451 expression by directly binding to its promoter region and, on its turn, is negatively regulated by miR-451. The expression of miR-144/451 is commonly down-regulated in B-lymphomas, and therefore, the axis MYC -miR-144/451generates a positive feedback loop which sustains the persistently elevated high MYC levels, in B-lymphocytes. By using a mouse model knocked out for miR-144/451 gene (miR-144/451 −/− ), it has been shown that the loss of miR-144/451 is important to initiate and promote B-lymphomagenesis through the subsequent activation of MYC [104]. Collectively, these observations highlight the broad impact of MYC-mediated miRNA reprogramming on cellular survival and proliferation pathways, through controlling apoptosis.

Conclusions and Remarks
Compromised apoptotic signaling promote tumorigenesis, particularly together with oncogenic lesions that drive excess cell proliferation [105,106]. MYC gene alterations have been identified in B-cell neoplasms, and are usually associated with aggressive clinical behavior. Development of novel techniques in the field of immunology and new animal models have greatly accelerated our understanding of oncogenic mechanisms in these MYC-associated lymphomas. Mouse models are a powerful tool to reveal multiple genes implicated in the genesis of lymphoma and are extensively used to clarify the molecular mechanism of lymphoma, validating the gene function. MYC is a transcription factor that regulates several relevant biological cellular events. Here, we have highlighted the transcriptional regulation network of MYC during lymphomagenesis, the molecular mechanisms of MYC collaborations that lead to escape from apoptosis, and the circuits between MYC and miRs involved in the pathogenesis of B-cell lymphomas harboring MYC alterations. Moreover, from the examples discussed above, the scientific evidence is increasing on novel interactors of MYC. These cooperations are at the head of a cascade that ultimately triggers apoptosis pathways. The dynamic interactions between anti-and pro-apoptotic proteins and their regulation by oncogene MYC depicted in Figure 1, have uncovered novel opportunities for therapeutic intervention. Therefore, new agents designed to specifically target MYC and its interactors will need to be improved. MYC-driven pathways are extremely intricate, involving a huge number of interactors, and affecting several cellular processes such as cell survival, proliferation, differentiation, and apoptosis, sometimes conflicting with each other. A deeper understanding of the molecular mechanisms of MYC roles and its interactors in each B-cell lymphoma types provides a wide set of candidates for the development of a better personalized targeted therapy that in near future could lead to successful treatments with less toxicity.