Small cell lung cancer (SCLC) is a deadly cancer type with a 6% five-year survival rate after diagnosis. However, the genetic events that dictate SCLC growth are not fully understood. In several models of SCLC, mutations and deletions have been identified in the transcription factor MAX, suggesting that MAX might have tumor suppressor activity. This finding presents a paradox: MAX binds to MYC oncoproteins to promote SCLC. Researchers in the MacPherson and Eisenman labs, led by postdoctoral fellows Dr. Arnaud Augert and Dr. Haritha Mathsyaraja, developed a CRISPR/Cas screen to identify candidate tumor suppressors genes and pathways in SCLC and identified MAX as a top hit in the screen. Further validation in cellular and in vivo models characterized MAX as a bonafide context-dependent tumor suppressor in SCLC. These results were recently published in the journal Cancer Cell.
The CRISPR/Cas9 screen was performed in preSC cells, an immortal cell line derived from early lesions in an SCLC mouse model. The screen targeted more than 20,000 mouse protein-coding genes and more than 1,000 microRNAs using a pooled library of mouse gRNAs packaged into a lentivirus that integrates into the target cell. In this system, the gRNAs that knock out a tumor suppressor will be overrepresented after the edited cells expand relative to an earlier reference time point. A CRISPR score can be calculated from the average fold change in abundances of all gRNAs targeting a given gene at final relative to initial time point. "Using genome-wide CRISPR/Cas9 screens, we were able to identify candidate tumor suppressor genes and pathways in SCLC. Surprisingly we identified MAX, an obligate heterodimerization partner for MYC, as a top hit in our screens," says Dr. Augert.
To validate their findings, the investigators depleted MAX in preSC cells, which resulted in increased cell proliferation and transformation. This effect was not observed in non-SCLC cells upon MAX depletion, suggesting that MAX’s tumor suppressor role is cell-type-specific. Next, the investigators sought to determine if MAX functions as an SCLC tumor suppressor in vivo. To this end, they developed a tissue-specific MAX knockout mouse model sensitized for SCLC. In this model, the depletion of MAX resulted in an increase in tumor volume and number of tumors in the lung, along with a decrease in survival rate. "Rigorous in vivo analyses using mouse models demonstrate a bona fide tumor suppressor role for MAX. Importantly, this work has generated, for the first time, an experimental system that will permit exploration of MAX’s tumor-suppressive functions", says Dr. Augert.
Using this tissue-specific MAX knockout model, the investigators set out to explain the MAX paradox: MAX supports tumor formation driven by MYCL, a member of the MYC oncogene family, while it acts as a tumor suppressor in the same type of tumors. When they overexpressed MYCL in the context of MAX loss, they saw that tumor cells overexpressing MYCL were under selection to retain MAX protein expression. Dr. Mathsyaraja explains these results: "Our experiments show that when MYC is present at high levels during SCLC progression, MAX must be present. However, tumor development in the absence of MAX appears to be independent of MYC, suggesting a novel pathway leading to SCLC growth." Thus, even though both MYCL overexpression and MAX-deletion cause cancer, the transformation events are mutually exclusive. The investigators then analyzed how MAX deletion affected transcription in three different SCLC cellular models and identified common genes differentially expressed across all models. Interestingly, they found enrichment for genes and networks involved in serine biosynthesis and one-carbon metabolism, pathways that support the high proliferative rate of cancer cells. Taken together, this body of work identifies MAX as a context-dependent tumor suppressor and establishes novel cellular and in vivo models to study MAX-driven gene regulation in SCLC.
This research was supported by grants from the National Institutes of Health and the National Cancer Institute.
UW/Fred Hutch Cancer Consortium members Dr. David MacPherson and Dr. Robert Eisenman contributed to this work.
Augert A, Mathsyaraja H, Ibrahim AH, Freie B, Geuenich MJ, Cheng PF, Alibeckoff SP, Wu N, Hiatt JB, Basom R, Gazdar A, Sullivan LB, Eisenman RN, MacPherson D. (2020). MAX Functions as a Tumor Suppressor and Rewires Metabolism in Small Cell Lung Cancer. Cancer Cell DOI: 10.1016/j.ccell.2020.04.016