MYC can’t cause havoc in B cells without MAX

From the Eisenman lab, Basic Sciences Division

The MYC oncogene family plays an important role in neoplastic disease, and has been extensively studied over decades. Evidence showing the N-terminal and C-terminal regions of MYC can function as a transcription activation domain and a dimerization and DNA binding domain respectively, has led to the assumption that MYC proteins would form homo or heterodimers, bind DNA and function as transcriptional activators.  MAX has been shown to interact specifically with all MYC family proteins; the resulting heterocomplexes recognize the hexameric DNA sequence CACGTG, part of the class of sequences known as E-boxes.  Studies from Dr. Robert Eisenman’s group in the Basic Sciences division have shown that MAX heterodimerization is required for MYC to bind to DNA, and carry out transcriptional and cell transformation activities. Nevertheless, there is also evidence that loss of MAX can be tolerated and sometimes oncogenic in certain biological contexts, suggesting that MYC can function independently of MAX to drive tumorigenesis.

Dr. Haritha Mathsyaraja, a postdoc in the Eisenman lab, took the lead in examining a MAX-independent role in MYC-driven oncogenesis. Together with other members of the Eisenman lab, along with members of the Henikoff lab, the authors show new insights into the molecular underpinnings of MYC regulation and cancer, and published this work in a recent issue of Genes & Development.

Dr. Mathsyaraja explains the importance of their finding: “MYC is a potent oncogene that’s amplified across a wide spectrum of malignancies. MAX was long thought to be an obligate hetero-dimerization partner for MYC in driving neoplasia, but no in vivo evidence existed to confirm that hypothesis. We for the first time conclusively show that MAX is required for MYC driven lymphomagenesis in vivo. In addition, we show that loss of MAX results in destabilization of MYC and a sharp reduction in MYC levels.”

Model depicting proposed network dynamics in normal B cells and pre-malignant Eµ-Myc cells and the consequences of Max deletion in each context.
Model depicting proposed network dynamics in normal B cells and pre-malignant Eµ-Myc cells and the consequences of Max deletion in each context. In normal B cells, MNT-MAX activity is higher than MYC-MAX activity, leading to the activation of a subset of MYC target genes. Upon Max loss, alleviation of MNT-MAX repression and E2F activation of target genes partially compensates for loss of MYC-MAX activity. In premalignant cells, MYC-MAX heterodimers show increased activity and activate MYC stabilizing genes such as Cip2a and Set. Disruption of this circuit via Max deletion leads to destabilization of MYC protein and loss of MYC signature expression. Hence, no tumors arise in KO mice. Figure from publication

The authors used the Eμ-Myc transgenic mice, which model the 8;14 translocation found in Burkitt’s B-cell lymphoma. To investigate the requirement of endogenous MAX in MYC-induced tumorigenesis, the authors generated a conditional Max allele to elucidate MAX function in lymphomagenesis and in B-cell homeostasis. Using this system, the authors found that Max deletion impairs B-cell development. Similarly, Eμ-Myc lymphomagenesis was also completely abolished in the absence of Max. RNA sequencing was then performed to determine the effects of Max loss on the transcriptional program of normal and Eμ-Myc expressing B cells. The results showed that Max loss affected E2F transcription factor targets and proinflammatory pathways in B cells. Genomic occupancy analysis using CUT&RUN (cleavage under targets and release using nuclease) was then performed on Max wild-type and knockout B cells, to reveal broad changes in genomic occupancy upon Max inactivation .

When the authors measured MYC levels in Max knockout B cells, they were surprised to see a striking reduction in MYC protein levels, while Myc mRNA expression remained unaffected. This influence of MAX on the stability of MYC held true in both normal and premalignant contexts. Notably, a small molecule inhibitor of MYC-MAX heterodimerization used on human B-cell lymphoma and several other cancer cell lines showed a decrease in MYC protein expression, reduced proliferation and proteins that support the stability of MYC.

Even though MYC has been extensively studied, finding therapeutic vulnerabilities has been challenging. Thus these findings are highly significant, as Dr. Mathsyaraja explains: “Several MYC-MAX dimerization inhibitors are currently in various stages of development as a therapeutic avenue for MYC driven tumors. Our work strongly suggests that such inhibitors would be doubly efficacious, targeting both MYC driven transcription and MYC protein levels. This is especially important considering that MYC can have MAX independent/ transcription independent functions.”

This work represents the first in vivo study showing that MYC requires MAX for tumorigenesis, through stabilizing MYC protein.  Dr. Mathsyaraja reveals what is next to come: One of the broader aims of our work is to elucidate the context dependent functions of MAX. MAX can function as a co-activator with MYC and promote tumorigenesis but at the same time it also dimerizes with MXD proteins to repress the same set of genes. We reasoned that under certain circumstances, MAX can act as a tumor suppressor. Indeed, reports suggest that MAX is mutated in certain neuroendocrine tumors including Small Cell Lung Cancer (SCLC). In collaboration with the MacPherson Lab, we are actively testing this hypothesis in a mouse model of SCLC.” 

Mathsyaraja H, Freie B, Cheng PF, Babaeva E, Catchpole JT, Janssens D, Henikoff S, Eisenman RN. 2019. Max deletion destabilizes MYC protein and abrogates Eµ-Myc lymphomagenesis. Genes Dev Sept 1; 33(17-18): 1252-1264.

This work was supported by the National Institutes of Health.

Cancer Consortium members Drs. Eisenman and Henikoff contributed to this research.