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Aberrant RAS/MAPK signaling is a common driver of oncogenesis that can be therapeutically targeted with clinically approved MEK inhibitors. Disease progression on single-agent MEK inhibitors is common, however, and combination therapies are typically required to achieve significant clinical benefit in advanced cancers. Here we focused on identifying MEK inhibitor-based combination therapies in neuroblastoma with mutations that activate the RAS/MAPK signaling pathway, which are rare at diagnosis but frequent in relapsed neuroblastoma. A genome-scale CRISPR-Cas9 functional genomic screen was deployed to identify genes that when knocked out sensitize RAS-mutant neuroblastoma to MEK inhibition. Loss of either CCNC or CDK8, two members of the mediator kinase module, sensitized neuroblastoma to MEK inhibition. Furthermore, small-molecule kinase inhibitors of CDK8 improved response to MEK inhibitors in vitro and in vivo in RAS-mutant neuroblastoma and other adult solid tumors. Transcriptional profiling revealed that loss of CDK8 or CCNC antagonized the transcriptional signature induced by MEK inhibition. When combined, loss of CDK8 or CCNC prevented the compensatory upregulation of progrowth gene expression induced by MEK inhibition. These findings propose a new therapeutic combination for RAS-mutant neuroblastoma and may have clinical relevance for other RAS-driven malignancies. Significance: Transcriptional adaptation to MEK inhibition is mediated by CDK8 and can be blocked by the addition of CDK8 inhibitors to improve response to MEK inhibitors in RAS-mutant neuroblastoma, a clinically challenging disease.

NXT2 expression is associated with NXT1 dependency

PDF file - 107KB, Two class comparison between combination treated samples and controls for the other GLUT proteins and hexokinses. The log2 of fold (combination/control) expression for each gene in the GLUT and hexokinase family are shown, such that negative values indicate a decrease in the combination relative to control, and positive values represent an increase. The p-value for the difference in expression between classes is shown.

CRISPR guide sequences

Differential NXT2 expression in neuroblastoma cell lines

PDF file - 47KB, Supplementary Figure 1 shows that AKT inhibition does not slow proliferation in a human MPNST cell line.

Supplementary Figure S1. MPNSTs are sensitive to combined HDAC and mTOR inhibtion; Supplementary Figure S2. Change in animal weight while on combiniation treatment; Supplementary Figure S3. A) 90-8TL cells were treated with Nexturastat A at concentration indicated, with (black) or without (white) 100nM sapanisertib; Supplementary Figure S4. Combined HDAC and mTOR inhibition increases reactive oxygen species in MPNSTs;Supplementary Figure S5. TXNIP is required for combination induced MPNST cell death;Supplementary Table S1. Differentially expressed gene sets in MPNSTs treated with HDAC and mTOR inhibitors

NXT1 is a selective and lethal dependency in neuroblastoma

NXT2 expression is not associated with neuroblastoma disease subtypes

PDF file - 75KB, Reported in vitro isoform specificities of PI3K inhibitors. The IC50 (nM) reported in the literature for each PI3K inhibitor against Class IA catalytic isoforms in in vitro binding assays is shown. The source for each data set is listed. A66-(S) is termed a p110alphaspecific inhibitor, AZD-6482 is classified as a p110beta-specific inhibitor, and CAL-101 shows selectivity for p110delta. GDC-0941 does not show significant isoform selectivity.

PDF file - 58KB

CRISPR screens prioritize rapidly lethal in vivo relevant dependencies

NXT2 loss leads to loss of NXF1

Cancer dependency maps, which use CRISPR/Cas9 depletion screens to profile the landscape of genetic dependencies in hundreds of cancer cell lines, have identified context-specific dependencies that could be therapeutically exploited. An ideal therapy is both lethal and precise, but these depletion screens cannot readily distinguish between gene effects that are cytostatic or cytotoxic. Here, we use a diverse panel of functional genomic screening assays to identify NXT1 as a selective and rapidly lethal in vivo relevant genetic dependency in MYCN-amplified neuroblastoma. NXT1 heterodimerizes with NXF1, and together they form the principal mRNA nuclear export machinery. We describe a previously unrecognized mechanism of synthetic lethality between NXT1 and its paralog NXT2: their common essential binding partner NXF1 is lost only in the absence of both. We propose a potential therapeutic strategy for tumor-selective elimination of a protein that, if targeted directly, is expected to cause widespread toxicity.Significance:We provide a framework for identifying new therapeutic targets from functional genomic screens. We nominate NXT1 as a selective lethal target in neuroblastoma and propose a therapeutic approach where the essential protein NXF1 can be selectively eliminated in tumor cells by exploiting the NXT1–NXT2 paralog relationship.See related commentary by Wang and Abdel-Wahab, p. 2129.This article is highlighted in the In This Issue feature, p. 2113

Supplemental Figures S1-S6 and Legends

Supplementary Table S1 STARS Scores

Aberrant RAS/MAPK signaling is a common driver of oncogenesis that can be therapeutically targeted with clinically approved MEK inhibitors. Disease progression on single-agent MEK inhibitors is common, however, and combination therapies are typically required to achieve significant clinical benefit in advanced cancers. Here we focused on identifying MEK inhibitor-based combination therapies in neuroblastoma with mutations that activate the RAS/MAPK signaling pathway, which are rare at diagnosis but frequent in relapsed neuroblastoma. A genome-scale CRISPR-Cas9 functional genomic screen was deployed to identify genes that when knocked out sensitize RAS-mutant neuroblastoma to MEK inhibition. Loss of either CCNC or CDK8, two members of the mediator kinase module, sensitized neuroblastoma to MEK inhibition. Furthermore, small-molecule kinase inhibitors of CDK8 improved response to MEK inhibitors in vitro and in vivo in RAS-mutant neuroblastoma and other adult solid tumors. Transcriptional profiling revealed that loss of CDK8 or CCNC antagonized the transcriptional signature induced by MEK inhibition. When combined, loss of CDK8 or CCNC prevented the compensatory upregulation of progrowth gene expression induced by MEK inhibition. These findings propose a new therapeutic combination for RAS-mutant neuroblastoma and may have clinical relevance for other RAS-driven malignancies.Significance:Transcriptional adaptation to MEK inhibition is mediated by CDK8 and can be blocked by the addition of CDK8 inhibitors to improve response to MEK inhibitors in RAS-mutant neuroblastoma, a clinically challenging disease.

Supplementary Table S3 Gene Sets

Cancer dependency maps, which use CRISPR/Cas9 depletion screens to profile the landscape of genetic dependencies in hundreds of cancer cell lines, have identified context-specific dependencies that could be therapeutically exploited. An ideal therapy is both lethal and precise, but these depletion screens cannot readily distinguish between gene effects that are cytostatic or cytotoxic. Here, we use a diverse panel of functional genomic screening assays to identify NXT1 as a selective and rapidly lethal in vivo relevant genetic dependency in MYCN-amplified neuroblastoma. NXT1 heterodimerizes with NXF1, and together they form the principal mRNA nuclear export machinery. We describe a previously unrecognized mechanism of synthetic lethality between NXT1 and its paralog NXT2: their common essential binding partner NXF1 is lost only in the absence of both. We propose a potential therapeutic strategy for tumor-selective elimination of a protein that, if targeted directly, is expected to cause widespread toxicity. Significance: We provide a framework for identifying new therapeutic targets from functional genomic screens. We nominate NXT1 as a selective lethal target in neuroblastoma and propose a therapeutic approach where the essential protein NXF1 can be selectively eliminated in tumor cells by exploiting the NXT1–NXT2 paralog relationship. See related commentary by Wang and Abdel-Wahab, p. 2129. This article is highlighted in the In This Issue feature, p. 2113

Mutations in the RAS/MAPK pathway are frequent drivers of oncogenesis. Clinically approved MEK inhibitors are effectively used to target RAS-pathway driven cancers in combination with RAF inhibitors in BRAF mutant cancers such as melanoma and non-small cell lung cancer. However, single agent treatment with MEK inhibitors is not typically sufficient for clinical benefit. Therefore, combination therapy approaches will be required for maximal efficacy in most advanced cancers. Here, we sought to identify MEK inhibitor-based combination therapy approaches in the setting of RAS-mutant neuroblastoma. Neuroblastoma is a cancer that arises in the developing peripheral nervous system. Children with low-risk disease frequently benefit from therapy, but 50-60% of children with high-risk disease will ultimately relapse, and relapsed disease is generally incurable. We performed genome-scale functional genomic drug modifier screens and identified mediator kinase module members CCNC and CDK8 as two genes that when lost sensitize RAS-mutant NBL to MEK inhibition. We used small molecule inhibitors of CDK8 and found that combined inhibition of MEK and CDK8 leads to an improved therapeutic response in vitro and in vivo in a xenograft model of neuroblastoma. Using transcriptional profiling, we established that CDK8 loss and MEK inhibition induce opposite transcriptional signatures. When combined, CDK8 loss prevents the compensatory upregulation of the pro-growth gene expression program induced by MEK inhibition, while the downregulation of MAPK signaling is maintained. Finally, we show that this combination is effective in a subset of other RAS-mutant cancers including pancreatic and lung cancers. Together these data suggest that the mediator kinase module is critical for the adaptive pro-survival transcriptional response induced by MEK inhibition in RAS-mutant neuroblastoma and other RAS-driven malignancies, and that the addition of a CDK8 inhibitor may improve clinical response to MEK inhibitors. Our data supports a model where agents with antagonistic transcriptional programs can have synergistic therapeutic effects when combined, which may be a broadly relevant approach in combination therapies. Citation Format: Clare F. Malone, Minjee Kim, Gabriela Alexe, Alexandra B. Forman, Amanda Robichaud, Amy Saur Conway, Amy Goodale, John M. Hatcher, Nathanael S. Gray, Federica Piccioni, Kimberly Stegmaier. Transcriptional antagonism by CDK8 inhibition improves therapeutic efficacy of MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2010.

NF1 encodes a RAS GTPase-activating protein. Accordingly, aberrant RAS activation underlies the pathogenesis of NF1-mutant cancers. Nevertheless, it is unclear which RAS pathway components represent optimal therapeutic targets. Here, we identify mTORC1 as the key PI3K effector in NF1-mutant nervous system malignancies and conversely show that mTORC2 and AKT are dispensable. However, we find that tumor regression requires sustained inhibition of both mTORC1 and MEK. Transcriptional profiling studies were therefore used to establish a signature of effective mTORC1–MEK inhibition in vivo. We unexpectedly found that the glucose transporter GLUT1 was potently suppressed, but only when both pathways were inhibited. Moreover, unlike VHL- and LKB1-mutant cancers, reduction of 18F-FDG uptake required the suppression of both mTORC1 and MEK. Together, these studies identify optimal and suboptimal therapeutic targets in NF1-mutant malignancies and define a noninvasive means of measuring combined mTORC1–MEK inhibition in vivo, which can be readily incorporated into clinical trials. Significance: This work demonstrates that mTORC1 and MEK are key therapeutic targets in NF1-mutant cancers and establishes a noninvasive biomarker of effective, combined target inhibition that can be evaluated in clinical trials. Cancer Discov; 4(9); 1062–73. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973

Pediatric cancers, such as neuroblastoma, have a relative lack of oncogenic mutations and the known drivers of these cancers are largely transcription factors, a target class notoriously difficult to “drug.” In order to identify novel therapeutic targets for high-risk, MYCN-amplified neuroblastoma, we employed functional genomic screening using a CRISPR-Cas9 approach. We generated genome-scale CRISPR dependency data to identify a candidate gene list of 197 putative genetic dependencies in neuroblastoma. We next created a focused sgRNA library targeting these genes and performed time-course dropout screens using CRISPR and CRISPRi and Annexin-V-based positive-selection cell death screens in four MYCN-amplified neuroblastoma cell lines. We also screened this library in vivo in two xenograft models of MYCN-amplified neuroblastoma. At the intersection of these screens, we identified the nuclear export factor NXT1 as a top candidate for therapeutic development in neuroblastoma. NXT1 scores as a strong dependency using both CRISPR and CRISPRi, is strongly enriched in our Annexin-V based positive selection cell death screen and scores in our in vivo screen. We have validated that NXT1 is indeed a genetic dependency in neuroblastoma in low-throughput and that NXT1 loss induces apoptosis in these cell lines. We were next interested in identifying biomarkers of dependency on NXT1, and by integrating RNAseq data with CRISPR dependency data, we identified low expression of NXT2, a paralog of NXT1, as the top predictive feature of NXT1 dependency. Importantly, while NXT1 itself is not currently druggable, inhibitors against another nuclear export protein, CRM1, are currently in Phase II clinical trials, demonstrating this class of proteins has the potential to be effectively drugged. Together, we show that CRISPR-Cas9 functional screens can be used to identify new therapeutic targets, particularly in diseases that lack “druggable” oncogenic drivers, such as many pediatric cancers. Citation Format: Clare F. Malone, Neekesh V. Dharia, Guillaume Kugener, Brenton Paolella, Michael Rothberg, Mai Abdusamad, Alfredo Gonzalez, Nancy Dumont, Scott Younger, David Root, Francisca Vazquez, Kimberly Stegmaier. CRISPR-Cas9 screens identify the nuclear export factor NXT1 as a novel therapeutic target in MYCN-amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2878.

Many children with metastatic or recurrent pediatric solid tumors continue to have poor survival, and there is an immense need to identify novel therapeutic approaches. Moreover, these cancers typically have simple genomes with limited known druggable molecular events. In order to discover new vulnerabilities in pediatric solid tumors, we have performed genome-scale CRISPR-Cas9 loss-of-function screening and deep “omic” characterization in over 60 pediatric cancer cell lines to date, including neuroblastoma, medulloblastoma, Ewing sarcoma, malignant rhabdoid tumor and rhabdomyosarcoma lines, to begin defining a pediatric cancer dependency map. Global analyses of the pediatric dependency landscape have identified emerging classes of pediatric cancers, including epigenetic-driven, aberrant transcription factor-driven and receptor tyrosine kinase-driven malignancies. For example, the preferential dependencies identified in a subset of neuroblastoma, which has aberrantly high expression of the transcription factor MYCN, are highly enriched for an interconnected network of genes annotated to have transcription factor activity. In addition to the global evaluation, we have developed methods and tools for prioritizing targets for further validation within a cancer type. These tools computationally integrate the pediatric dependency data across multiple datasets to identify categories of genetic dependencies that are especially strong hits or enriched hits in a specific pediatric malignancy. As an example, the intersection of MYCN-amplified neuroblastoma specific dependencies and H3-lysine 27 acetylation (H3K27ac) profiling across MYCN-amplified neuroblastoma allowed us to identify a transcriptional core regulatory circuit (CRC) that may drive the malignant state. Furthermore, targeting transcription with the BRD4 inhibitor JQ1 and CDK7 inhibitor THZ1 caused synergistic killing of neuroblastoma cells suggesting a novel therapeutic approach to treating this disease. Thus, defining a comprehensive pediatric cancer dependency map and developing the methods and tools to prioritize vulnerabilities in different cancer types will allow us to discover both novel biology and new therapeutic opportunities in childhood malignancies. Citation Format: Neekesh V. Dharia, Clare Malone, Amanda Balboni Iniguez, Lillian Guenther, Liying Chen, Gabriela Alexe, Adam D. Durbin, Mark W. Zimmerman, Andrew Hong, Pratiti Bandopadhayay, Mariella G. Filbin, Thomas Howard, Brenton Paolella, Iris Fung, Josephine Lee, Phil Montgomery, John M. Krill-Burger, Brian J. Abraham, Jennifer Roth, David E. Root, Richard A. Young, A. Thomas Look, Rameen Beroukhim, Jesse S. Boehm, William C. Hahn, Todd R. Golub, Aviad Tsherniak, Francisca Vazquez, Kimberly Stegmaier. Defining a pediatric cancer dependency map [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2352.