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MYCN amplification and overexpression are common in neuroendocrine prostate cancer (NEPC). However, the impact of aberrant N-Myc expression in prostate tumorigenesis and the cellular origin of NEPC have not been established. We define N-Myc and activated AKT1 as oncogenic components sufficient to transform human prostate epithelial cells to prostate adenocarcinoma and NEPC with phenotypic and molecular features of aggressive, late-stage human disease. We directly show that prostate adenocarcinoma and NEPC can arise from a common epithelial clone. Further, N-Myc is required for tumor maintenance, and destabilization of N-Myc through Aurora A kinase inhibition reduces tumor burden. Our findings establish N-Myc as a driver of NEPC and a target for therapeutic intervention.

Purpose: Alteration of the BRAF/MEK/MAPK pathway is the hallmark of pediatric low-grade gliomas (PLGGs), and mTOR activation has been documented in the majority of these tumors. We investigated combinations of MEK1/2, BRAFV600E and mTOR inhibitors in gliomas carrying specific genetic alterations of the MAPK pathway. Experimental Design: We used human glioma lines containing BRAFV600E (adult high-grade: AM-38, DBTRG, PLGG: BT40), or wild-type BRAF (pediatric high-grade: SF188, SF9427, SF8628) and isogenic systems of KIAA1549:BRAF-expressing NIH/3T3 cells and BRAFV600E-expressing murine brain cells. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Proliferation was determined using ATP-based assays. In vivo inhibitor activities were assessed in the BT40 PLGG xenograft model. Results: In BRAFV600E cells, the three possible doublet combinations of AZD6244, everolimus, and PLX4720 exhibited significantly greater effects on cell viability. In BRAFWT cells, everolimus + AZD6244 was superior compared with respective monotherapies. Similar results were found using isogenic murine cells. In KIAA1549:BRAF cells, MEK1/2 inhibition reduced cell viability and S-phase content, effects that were modestly augmented by mTOR inhibition. In vivo experiments in the BRAFV600E pediatric xenograft model BT40 showed the greatest survival advantage in mice treated with AZD6244 + PLX4720 (P < 0.01). Conclusions: In BRAFV600E tumors, combination of AZD6244 + PLX4720 is superior to monotherapy and to other combinatorial approaches. In BRAFWT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition. Application of these results to PLGG treatment must be exercised with caution because the dearth of PLGG models necessitated only a single patient-derived PLGG (BT40) in this study. Clin Cancer Res; 22(21); 5312–21. ©2016 AACR.

Pediatric low-grade gliomas (PLGG) constitute the most common group of central nervous system tumors in children. Viewed as a chronic disease, ideal therapy for PLGG should carry limited side effects that can be best achieved through selective, personalized therapy. Despite the known heterogeneity of PLGGs and characterized driver mutations most children are still treated with standard chemotherapy protocols as first line therapy. Alteration of the BRAF/MEK/MAPK pathway is the hallmark of PLGG and mTOR activation has been documented in the majority of these tumors. This study investigates combinations of MEK1/2, BRAFV600E and mTOR inhibition in PLGGs carrying specific genetic alterations of the MAPK pathway. We used human glioma cell lines containing BRAFV600E (AM38, DBTRG, BT40), or wild-type BRAF (SF188, SF9427, SF9402) and isogenic systems of KIAA1549:BRAF-expressing NIH3T3 cells and BRAFV600E-expressing murine brain tumors. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), and AZD6244 (MEK1/2). Cell cycle distribution and apoptosis were assessed using flow cytometry; proliferation was determined using an ATP-based assay (CellTiter-Glo). In vivo activity of these inhibitors was assessed in the BT40 PLGG xenograft mouse model. In BRAFV600E tumors, the combination of MEK inhibitor with either mTOR or BRAFV600E specific inhibitor is superior to monotherapy and to combination of BRAF and mTOR inhibitors in reducing cell viability reduction and arresting cell cycle progression. In BRAFWT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition alone. In vivo studies in the PLGG xenograft model BT40 showed the greatest survival advantage in mice treated with combination of AZD6244 + PLX4720 or AZD6244 + everolimus compared with respective monotherapies (p < 0.01).

SummaryMYC proteins are major drivers of cancer yet are considered undruggable because their DNA binding domains are composed of two extended alpha helices with no apparent surfaces for small-molecule binding. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora A. We describe a class of inhibitors that disrupts the native conformation of Aurora A and drives the degradation of MYCN protein across MYCN-driven cancers. Comparison of cocrystal structures with structure-activity relationships across multiple inhibitors and chemotypes, coupled with mechanistic studies and biochemical assays, delineates an Aurora A conformation-specific effect on proteolytic degradation of MYCN, rather than simple nanomolar-level inhibition of Aurora A kinase activity.

The concept that some childhood malignancies arise from postnatally persistent embryonal remnant or rest cells has a long history. Recent research has strengthened the links between driver mutations, and, embryonal and early postnatal development. This evidence, coupled with much greater detail on the cell of origin and the initial steps in embryonal cancer initiation, has identified important therapeutic targets and provided renewed interest in strategies for the early detection and prevention of childhood cancer.

MYC family proteins represent a critical node in oncogenesis across a wide range of pediatric and adult cancers. This is particularly true in neuroblastoma, where amplification of MYCN confers an especially poor prognosis. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora Kinase A. We describe a class of allosteric inhibitors that disrupts the native conformation of Aurora A and causes degradation of MYCN protein across MYCN-expressing neuroblastoma cell lines. We compare co-crystal structures with structure-activity relationships across multiple inhibitors and chemotypes to delineate an Aurora A conformation-specific effect on proteolytic degradation of MYCN protein, which is driven markedly by conformation disrupting inhibitors, and impacted more modestly by current clinical Aurora Kinase A inhibitors, which disrupt the kinase structure less efficiently. Using functional assays of apoptosis, cell cycle, and proliferation we further define a distinct MYCN-specific effect distinguishing it from simple inhibition of mitosis by conventional Aurora kinase inhibitors. This new class of conformation disrupting inhibitors, which block stabilizing interactions between Aurora A and MYCN, represents a novel strategy to target MYCN-driven cancers. This abstract is also presented as Poster B46. Citation Format: W. Clay Gustafson, Justin G. Meyerowitz, Erin A. Nekritz, Elise Charron, Katherine K. Matthay, Nicholas T. Hertz, Martin Eilers, Kevan M. Shokat, William A. Weiss. Drugging MYCN protein stability through an allosteric transition in Aurora kinase A. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR08.

Amplification of MYCN is associated with high-risk SHH-driven and group 4 medulloblastoma, while mis-expression and amplification of MYC is associated with aggressive group 3 tumors. The biology of these tumors, particularly the group 3/4 tumors, is poorly understood, with no effective targeted therapies. We have recently developed a model of group 4 medulloblastoma in which high levels of mutationally-stabilized MYCN protein drive oncogenesis. We have shown in other tumor types that modulation of the translational apparatus downstream of the mammalian target of rapamycin (mTOR) is required for MYC-driven oncogenesis and that there is a synergistic relationship between MYC and the mTOR axis to promote tumor formation. mTOR signals through two primary outputs, rpS6 kinase (S6K) and the translation initiation factor eIF4E. A new class of clinical mTOR active site inhibitors disrupts signaling through both effectors, whereas the allosteric binder rapamycin disrupts only S6K. We describe genetic and therapeutic studies aimed at distinguishing the effects of S6K and eIF4E in both tumor formation and in therapy. These mechanistically distinct activities have enormous therapeutic implications, as in our medulloblastoma model, active site inhibitors of mTOR, but not rapamycin, demonstrate efficacy. Taken together, this supports the idea that signaling through S6K is dispensable, whereas eIF4E is required for MYC-driven oncogenesis. Citation Format: Justin G. Meyerowitz, W. Clay Gustafson, Erin Nekritz, Fredrik Swartling, Kevan M. Shokat, Davide Ruggero, William A. Weiss. Targeting the translational apparatus in MYCN-driven medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B78.

MYC genes contribute to a range of cancers including neuroblastoma, where amplification of MYCN confers a poor prognosis. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora kinase A. We describe a class of inhibitors that disrupts the native conformation of Aurora A and causes degradation of MYCN protein across MYCN-expressing neuroblastoma cell lines. Comparison of co-crystal structures with structure- activity relationships across multiple inhibitors and chemotypes, coupled with mechanistic studies and biochemical assays, delineates an Aurora A conformation-specific effect on proteolytic degradation of MYCN, rather than simple nanomolar-level inhibition of Aurora A kinase activity. This new class of inhibitors, which disrupts stabilizing interactions between Aurora A and MYCN, represents candidate agents to target MYCN-driven and potentially MYC-driven cancers. Citation Format: William C. Gustafson, Justin G. Meyerowitz, Erin A. Nekritz, Elise A. Charron, Yvan Chanthery, Erin Simonds, Katherine Matthay, Nicholas Hertz, Kevan Shokat, William A. Weiss. Drugging MYCN through an allosteric transition in Aurora A kinase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2688. doi:10.1158/1538-7445.AM2014-2688