The identification of agents that can elicit oncogene addiction has led to the successful treatment of cancer. The mechanism by which oncogene addiction occurs is not entirely clear, but appears to causally associated with the change in the balance of pro-survival and pro-death signals necessarily evoking tumor regression (16
). However, effective small-molecules inhibitors of established oncogenes are rare. Moreover, even if such agents could be identified the broad inhibition of an oncogene that also has normal functions could be highly toxic. Thus, a general strategy for identifying effective agents to target oncogenes and in particular to inhibit them in a manner that would elicit oncogene addiction but minimize toxic effects would be highly useful.
The targeted inactivation of MYC would be potentially useful for the treatment of many cancers. However, TFs are particularly difficult to target. A strategy for their context-specific inactivation would be generally valuable for many TF oncogenes. Moreover, given the pleiotropic regulatory properties of TFs, their systemic inactivation would likely affect many critical cellular functions, resulting in severe toxicity. Furthermore, TFs are often characterized as undruggable targets, due to scarcity of well-defined small-molecule binding pockets in their 3D structure. Thus, availability of methodologies to identify druggable TF modulators whose inhibition will abrogate TF activity in vivo could have important implications in designing targeted therapeutic strategies.
Here, we show that computationally predicted TF modulators, inferred by the MINDy algorithm (24
), can provide actionable upstream targets for inactivation of oncogenic TF-regulated programs within a given cellular context, which can abrogate tumorigenesis in vivo
Our results provide novel mechanistic insight into post-transcriptional regulation of MYC. We identify a new strategy for therapeutic targeting MYC in hematopoietic malignancies. Our results illustrate that STK38 is important in post-translational regulation of MYC’s function and may be a novel therapeutic target. The characterization of post-translational mechanisms for control of MYC’s transcriptional activity by STK38 suggest a more general role for its homeostatic regulation of normal cell physiology that, when deregulated, can contribute to tumorigenesis. Previous literature has correlated changes in STK38 expression with tumorigenesis. Some reports suggest that upregulation of STK38 is associated with progressive breast ductal carcinoma in situ (25
) and melanoma (26
). Conversely, significant downregulation of STK38 mRNA was shown in gastric cancer compared with normal gastric mucosa (27
) and B-cell lymphoma compared with normal B-cells (28
). Therefore, the mechanism by which STK38 deregulation contributes to tumorigenesis appears to be context dependent.
We show that STK38 plays a critical role in transduction of BCR signals in human Bcells and can modulate MYC activity by distinct kinase-activity-dependent mechanisms, including: (a) interaction with distinct MYC protein domains, (b) regulation of MYC protein turnover and (c) modulation of MYC transcriptional activity, independent of MYC protein levels. Given the range and significance of the various mechanisms by which STK38 modulates MYC activity, we will discuss them individually.
Post-translational modifications of MYC, including phosphorylation, ubiquitination, and acetylation affect protein stability and are critical effectors of MYC transcriptional activity (29
). Despite their significance, understanding the signal transduction cascades affecting MYC post-translational modification is still sparse. Previously, we demonstrated that STK38 kinase mediates MYC phosphorylation (3
). In this report we elucidate the kinase-dependent nature of STK38-MYC interaction. STK38 binds to the MYC C-terminal region (201–439 aa) which includes the basic DNA-binding helix-loop-helix-leucine zipper (b/HLH/LZ) domain (30
). Proteins known to interact with MYC in this region, including Max, BRCA1, AP-2, YY1, TFII and Miz-1, are all involved in regulation of MYC transcriptional activity (31
We directly demonstrate that STK38 regulates MYC’s transcriptional activity using a TERT-reporter in U2OS cells. Furthermore, GSEA analysis of STK38 silencing in ST486 cells line showed that STK38 regulates MYC transcriptional activity. Conversely, the kinase inactive form of STK38 had no effect on MYC transcriptional activity. This could be explained by its interaction with the internal MYC domain (145–353 aa) with a region called “MYC box III” (MbIII) previously shown to be indispensable for MYC transcriptional repression of target genes (32
) and its degradation (33
). Binding of kinase inactive STK38 to this region could interfere with both processes. Although, a transactivation domain (TAD) located in the MYC N-terminus is known to be involved in MYC transcriptional activity (34
) it did not interact with STK38 in our co-immunoprecipitation assay. Future investigation will determine whether regulation of MYC transcriptional activity by STK38 is due to differential binding of the kinase to MYC protein or engagement of other co-activating molecules. Recent studies have demonstrated post-translational regulation of MYC is, in part, mediated through coordination of regulatory proteins into complexes containing multi-domain scaffolding proteins (35
). Although speculative, it is worth considering that STK38 may be participating in the multi-protein degradation complex involved in MYC regulation.
Our data suggest that MYC turnover is regulated by STK38 activity, with active STK38 decreasing protein levels while inactive STK38 extends MYC half-life. The significant increase in MYC turnover following STK38 overexpression, as well as silencing, suggests that this kinase could provide a therapeutic target for MYC activity modulation in MYC-dependent tumors. This is provocative, since the direct pharmacological targeting of MYC is challenging. Others have reported that overexpression of active STK38 in 293T cells impaired MYC ubiquitination, although STK38 could not compete with FBW7 ubiquitin ligase for MYC binding (36
). Our results indicate that overexpression of active STK38 in B-cell context does not interfere with MYC degradation in contrast to inactive STK38. Whether reduction in MYC turnover in the presence of inactive STK38 results from inefficient proteasomal degradation due to either “protective” binding to the MYC internal domain or phosphorylation-dependent modulation of its ubiquitination, must be defined.
We identified the inverse correlation between MYC half-life and activity following increased expression of STK38-WT. Similar findings have been documented for other proteins (37
), using chimeric transcriptional activators (38
). Increased activity of the transcriptional activator correlated with enhanced proteasome-mediated degradation, and was one possible mechanism of modulation of intracellular levels of regulatory proteins. A similar mechanism was reported for ubiquitin ligase SKP2, which was shown to increase MYC transcriptional activity while decreasing its protein stability (39
We speculate that the multilayered regulation by STK38 can be explained by a number of regulatory mechanisms effecting intrinsically disordered proteins (IDPs) such as MYC (40
). Native unstructured regions present in many eukaryotic proteins (43
) are involved in transcriptional regulation and signaling events and their levels and accessibility must be tightly controlled. Unstructured regions of IDPs are susceptible to ubiquitin-independent degradation and post-translational modifications which allow precise regulation (44
). Such regulation can be mediated through binding to other proteins that effectively protect the IDP from degradation or mediate the IDP phosphorylation state (45
). In that context, inactive STK38 could play a role of a “nanny” (45
) by binding to the internal domain of MYC and protecting it from degradation. On the other hand, wild type structure of phosphorylated form of STK38 could favor binding to C-terminus of MYC allowing its full functionality.
We have shown that STK38 is a key effector of BCR-signaling-mediated, MYC-dependent apoptosis and proliferative arrest in human B-cells in concordance with our previous results (2
). Depending on its kinase activity, STK38 can either stabilize or destabilize MYC protein levels and affect MYC transcriptional activity. Operating within a complex post-translational regulatory landscape, STK38 overexpression may have opposite effects depending on its kinase activity. This is consistent with previously reported dual-role proteins, like p38 or JNK, which could contribute to both anti- and pro-oncogenic processes (46
). Similar view on the dual function of NDR1/2 kinases was discussed broadly in (47
STK38’s function in specific signaling pathways is poorly defined. So far it has only been characterized as a negative regulator of MEKK1/2, a member of MAPK signaling pathway by exogenous expression in 293T cells (7
) and a regulator of p21 stability in cell cycle progression (36
). We have defined a role of STK38 in BCR signaling. Our results illustrate that STK38 is a critical effector of BCR-mediated signals modulating MYC protein activity and turnover, as well as cell survival, suggesting that this kinase plays a prominent role in the BCR pathway. BCR activation mediated apoptosis was completely abrogated in cells expressing the kinase inactive form, STK38-KD. STK38 mediated regulation of apoptosis was demonstrated by others in HeLa cells, where transient overexpression of wild-type STK38 enhanced release of apoptotic markers (18
Concordant with the notion of oncogene addiction, the targeted inactivation of critical genes in oncogenic pathways that are directly responsible for the ability of an oncogene to maintain survival over death signaling would in turn lead to the regression of cancer (15
). Hence, targeting these co-dependent genes could elicit oncogene addiction identical to the suppression of the oncogene (48
). Therefore, we hypothesized that targeting a key regulator of MYC protein expression would lead to abrogation of MYC induced tumors. In vivo
mouse xenograft models demonstrated that silencing STK38 leads to delayed tumor formation and slowed tumor growth. At least two mechanisms underlie this process; decreased proliferation and increased apoptosis. These intrinsic mechanisms aid in the tumor regression observed in MYC inactivation models (23
). It appears that balance between pro-death and pro-life is shifted toward pro-death in tumors in which STK38 function is removed. Although, in STK38 knockdown tumors an increase in mass after 25 days of growth was observed, it is noteworthy that both STK38 and MYC protein levels were increased as well, suggesting that a subpopulation of cells within the tumor eventually found a way to circumvent IPTG-inducible loss of STK38. Although, we cannot rule out the possibility that the IPTG induction was not efficient in all cells within the tumor mass, it’s interesting to speculate that this observation indicates some bypass mechanism similar to those observed in the reversal of MYC tumor regression (49
Our results identify a new general strategy for the identification of key regulators of oncogenes that in turn can serve as therapeutic targets to elicit oncogene addiction. Specifically, we demonstrate that STK38 critically regulates the MYC protein and its activity (), at several cellular levels; 1) protein-protein interaction, 2) protein turnover, and 3) transcriptional activity. We show that STK38 is crucial for survival of the ability of MYC-induced lymphomas (15
). Hence, suppression of STK38 elicits oncogene addiction in MYC-addicted tumors. Thus, through the combined computational and experimental characterization of effectors of TF activity, we can isolate key upstream regulators of oncogenes, and in particular a TF such as MYC, that are critically required for these oncogenes to maintain a neoplastic phenotype. Therefore, this is a new paradigm for eliciting oncogene addiction, through the suppression of genes that are critical and co-dependent regulators of oncogenes, hence functioning as surrogate and more readily druggable therapeutic targets.