MYCN drives the development of chemoresistance in small cell lung cancer

From the MacPherson lab, Human Biology Division

Resistance to chemotherapy, known as chemoresistance, is a significant hurdle in cancer therapy. In small cell lung cancer (SCLC), an aggressive form of lung cancer, initial treatment is effective. Still, chemoresistance develops within a year, leading to ultimate treatment failure and eventually death. Thus, identifying SCLC chemoresistance drivers is of vital importance to improve the prognosis for SCLC patients. In cell lines derived from chemotherapy-treated patients, an increase MYC family member copy number, relative to samples from treatment-naïve individuals, suggests that the MYC family of transcription factors could play a role in the development of chemoresistance in SCLC.

Previous studies had demonstrated that the overexpression of MYC family members MYC and MYCL promotes SCLC in mice. However, the role of MCYN amplification in SCLC progression and chemoresistance remain poorly understood. New research from the MacPherson Lab (Human Biology Division) published in the journal Genes & Development identifies MYCN as a driver for chemoresistance in SCLC. In the study, the investigators developed the “RPMYCN” mouse, a novel autochthonous mouse model with an inducible MYCN-overexpression system. In the RPMYCN mouse, lung tumors grew faster than in the parental mouse strain “RP.” Magnetic resonance imaging (MRI) and histological analysis revealed that the tumors’ location and structure reflect classic human SCLC, making it a suitable model to study the role of MYCN overexpression in SCLC. 

Since MYCN expression is inducible by doxycycline (DOX) administration in the RPMYCN mouse model, the investigators asked if sustained MYCN expression is essential for SCLC tumors initiated with high levels of MYCN. Remarkably, upon DOX removal, tumors regressed by at least 25% in most of the mice. However, tumors eventually returned, demonstrating that while rapid tumor growth in RPMYCN mice is dependent on MYCN, tumors regain the ability to grow without sustained MYCN activation. Detection of the cell proliferation marker pH3 in DOX-treated cells indicated increased proliferation, and DOX removal led to increased cell death as determined by the detection of TUNEL-positive cells via immunohistochemistry. These results were validated in independent cell cultures derived from several RPMYCN mice. DOX removal from the growth medium led to reduced viability due to increased cell death and reduced N-MYC protein levels. On DOX, RPMYCN cell lines exhibited an increase in proliferation, protein synthesis, and functional inactivation of the potent tumor suppressor p130. Taken together, these results demonstrate that MYCN overexpression promotes proliferation and increases protein synthesis.

Next, the investigators characterized the transcriptional changes resulting from MYC overexpression. Using gene set enrichment analysis, they identified upregulation of MYC targets and members of the unfolded protein response. Interestingly, genes involved in immune signaling pathways were downregulated in MYCN overexpression conditions, suggesting that MYCN overexpression may alter the SCLC’s tumor immune microenvironment. Flow cytometry analysis on cells derived from RPMCYN on DOX, RPMYCN off DOX, and RP tumors showed an overall reduction of T cells and a higher percentage of neutrophils in RPMYCN on DOX. These results are of particular interest because tumor microenvironments with low T cell and high neutrophil infiltration are correlated with poor outcomes.

 To test if MYCN overexpression can confer chemoresistance, the investigators used MRI to monitor tumor volume in RP and RPMYCN mice after saline or chemotherapy drug cisplatin–etoposide (cis-eto). RPMYCN tumors continued to grow after treatment, whereas RP tumors had reduced cell growth and division compared to the saline-only treatment. Next, the instigators obtained similar results in an SCLC MYCL-overexpression mouse model (RPMYCL).  Taken together, these experiments showed that, in both RPMYCL and RPMYCN mice, overexpression of the MYC transgene could suppress the responses to chemotherapy. These findings were supported by the generation of patient-derived xenograft (PDX) mouse models implanted with chemosensitive SCLC tumors modified to express MYCN or MYCL. Upon MYC transgene expression, these tumors gained chemoresistance.

Finally, the investigators performed a genome-wide CRISPR-Cas9 screen to identify druggable genes associated with MYCN overexpression. They identified USP7; a gene encodes a deubiquitinase that directly targets N-MYC, resulting in increased protein stability. In vitro testing of a USP7 inhibitor (USP7i) showed an increase in apoptosis and reduced N-MYC protein. In vivo, MYCN tumors were responsive to a combination of cis-eto and USP7i. In summary, this work demonstrated that MYCN overexpression promoted accelerated SCLC, led to changes in the tumor immune microenvironment, and identified a druggable target to reduce N-MYC. 

This work was supported by grants from the National Institutes of Health.

Fred Hutch/UW Cancer Consortium members McGarry Houghton, and David MacPherson contributed to this work.

Grunblatt E, Wu N, Zhang H, Liu X, Norton JP, Ohol Y, Leger P, Hiatt JB, Eastwood EC, Thomas R, Ibrahim AH, Jia D, Basom R, Eaton KD, Martins R, Houghton AM, MacPherson D. 2020. MYCN drives chemoresistance in small cell lung cancer while USP7 inhibition can restore chemosensitivity. Genes Dev. 2020 Sep 1;34(17-18):1210-1226. doi: 10.1101/gad.340133.120. Epub 2020 Aug 20.