JulieAnn Rader

Dual CDK4/CDK6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma

Purpose: Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell-cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes. Experimental Procedures: We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011 (Novartis Oncology), a highly specific CDK4/6 inhibitor. Results: Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nmol/L in sensitive lines). LEE011 caused cell-cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. Although our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (P = 0.01), the identification of additional clinically accessible biomarkers is of high importance. Conclusions: Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. Clin Cancer Res; 19(22); 6173–82. ©2013 AACR.

Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations

The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway. Seven of these events were detected only in the relapse tumor, whereas the others showed clonal enrichment. In neuroblastoma cell lines, we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18; 61%), and these lesions predicted sensitivity to MEK inhibition in vitro and in vivo. Our findings provide a rationale for genetic characterization of relapse neuroblastomas and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.

Combination Therapy Targeting the Chk1 and Wee1 Kinases Shows Therapeutic Efficacy in Neuroblastoma

Neuroblastoma is uniquely sensitive to single-agent inhibition of the DNA damage checkpoint kinase Chk1, leading us to examine downstream effectors of this pathway and identify mitotic regulator Wee1 as an additional therapeutic target in this disease. Wee1 was overexpressed in both neuroblastoma cell lines and high-risk patient tumors. Genetic or pharmacologic abrogation of Wee1 signaling results in marked cytotoxicity in 10 of 11 neuroblastoma cell lines with a median IC(50) of 300 nmol/L for the Wee1-selective small-molecule inhibitor MK-1775. Murine tumor lines derived from mice that were either heterozygous or homozygous for MycN were particularly sensitive to single-agent inhibition of Wee1 (IC(50)s of 160 and 62 nmol/L, respectively). Simultaneous pharmacologic inhibition of Chk1 and Wee1 acted in a synergistic fashion to further impede neuroblastoma cell growth in vitro, in a manner greater than the individual inhibitors either alone or combined with chemotherapy. Combination Chk1 and Wee1 inhibition also revealed in vivo efficacy in neuroblastoma xenografts. Taken together, our results show that neuroblastoma cells depend on Wee1 activity for growth and that inhibition of this kinase may serve as a therapeutic for patients with neuroblastoma.

Dielectrophoretic Capture and Genetic Analysis of Single Neuroblastoma Tumor Cells

Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.

A novel approach for next-generation sequencing of circulating tumor cells.

Next‐generation sequencing (NGS) of surgically resected solid tumor samples has become integral to personalized medicine approaches for cancer treatment and monitoring. Liquid biopsies, or the enrichment and characterization of circulating tumor cells (CTCs) from blood, can provide noninvasive detection of evolving tumor mutations to improve cancer patient care. However, the application of solid tumor NGS approaches to circulating tumor samples has been hampered by the low‐input DNA available from rare CTCs. Moreover, whole genome amplification (WGA) approaches used to generate sufficient input DNA are often incompatible with blood collection tube preservatives used to facilitate clinical sample batching.

Preclinical Therapeutic Synergy of MEK1/2 and CDK4/6 Inhibition in Neuroblastoma

Purpose: Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition. Experimental Design: We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies. Results: Sensitivity to binimetinib and ribociclib was inversely related (r = −0.58, P = 0.009). MYCN amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs. Conclusions: Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS–MAPK signaling. Clin Cancer Res; 23(7); 1785–96. ©2016 AACR.

CASZ1 inhibits cell cycle progression in neuroblastoma by restoring pRb activity.

Dysregulation of cell cycle genes such as Cyclin D1 and Chk1 contributes to the undifferentiated phenotype of neuroblastoma (NB). CASZ1 functions as a tumor suppressor in NB; here we sought to determine how loss of CASZ1 contributes to cell cycle dysregulation in NB. CASZ1 restoration in NB cells delays NB cell cycle progression. The earliest changes occur within 8 h of CASZ1 restoration in SY5Y cells with a 2.8-fold increase in the level of p21, an inhibitor of Cdk2/4. By 16 h, there is a 40% decrease in the steady-state levels of Cdk6. Restoration of CASZ1 decreases Cdk2-dependent cyclins A and E protein levels and Cdk4/6-dependent Cyclin D1 protein levels. The restoration of CASZ1 resulted in a decrease in pRb phosphorylation and a significant reduction of E2F transcriptional activity. Subsequent to the changes in the G1/S transition, induction of CASZ1 results in a decrease in Cyclin B levels and Cdc25c phosphatase levels, an upstream activator of the G2/M regulator CyclinB:Cdk1. In addition, induction of CASZ1 results in a decrease in the levels of phospho-Chk1, a key M-phase regulatory kinase. Similar results were found in a NB cell line with MYCN amplification. Taken together, this study indicates that restoration of CASZ1 activates pRb in G1 and inhibits the G2/M regulators Cyclin B1 and Chk1, leading to a lengthening of NB cell cycle progression and a subsequent decrease in cell proliferation.

Abstract 2744: CDK4/CDK6 inhibition is potently active in a definable subset of human neuroblastomas.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Neuroblastoma is a pediatric embryonal cancer for which the survival of patients with high-risk disease is less than 50% and has not dramatically changed over the last several years. Recently, a number of cell cycle genes\_particularly those within the Cyclin D/CDK4/CDK6/RB network\_have been identified as oncogenic vulnerabilities in neuroblastoma, suggesting that their therapeutic exploitation might improve survivability. Indeed, genomic amplifications of CDK4, CDK6, and CCND1 have been reported in primary neuroblastomas, and we have previously shown via an unbiased loss of function screen that CDK4 depletion is associated with potent anti-tumor activity (Cole, PNAS 2011). Here, we sought to translate these findings into novel therapies for children with neuroblastoma by evaluating the effect of pharmacologic Cdk4/Cdk6 inhibition on neuroblastoma viability. Methods: We analyzed the effect of combined Cdk4/6 inhibition in a comprehensive panel of human-derived neuroblastoma cell lines using LEE011, a highly specific Cdk4/6 small molecule inhibitor. Anti-tumor activity was also determined in vivo in three neuroblastoma xenograft models, and integrative genomics was used to identify biomarkers of drug sensitivity. Results: Treatment with LEE011 significantly inhibited proliferation in 10 of 15 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 361 ± 97 nM, considering sensitive lines only), as evidenced by significant cell cycle arrest and senescence that were likely attributed to dose-dependent decreases in phosphorylated RB and FOXM1. In addition, responsiveness of neuroblastoma xenografts to LEE011 was reflective of in vitro data in that there was a direct correlation of IC50 values with degree of subcutaneous xenograft growth delay, with the most sensitive lines in vitro showing profound growth inhibition in vivo. While our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (p= 0.04, students t test), a supervised hierarchical clustering of gene expression data identified several potential gene signatures that could explain the observed differential sensitivity to Cdk4/6 inhibition. Conclusions: Our data show that LEE011 is highly active in a large subset of neuroblastoma cell lines and xenograft models, and therefore support the clinical development of LEE011 as a therapy for neuroblastoma as well as efforts to validate biomarkers of drug activity. Citation Format: JulieAnn Rader, Lori Hart, Mike Russell, Michael Nakazawa, Lili Belcastro, Daniel Martinez, Erica Carpenter, Sunkyu Kim, Sudha Parasuraman, Giordano Caponigro, Robert Schnepp, Andrew Wood, Bruce Pawel, Deborah Watson, Patrick Warren, Kristina Cole, John Maris. CDK4/CDK6 inhibition is potently active in a definable subset of human neuroblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2744. doi:10.1158/1538-7445.AM2013-2744

Abstract LB-126: The identification of synthetic lethal partners cooperating with MEK1/2 inhibition in neuroblastoma

Background: Neuroblastoma, a pediatric malignancy of the developing sympathetic nervous system, is treated with aggressive multi-modal therapy, yet more than 50% of patients experience relapses for which there are limited treatment options. Improvements in the management of relapsed neuroblastoma require the identification of novel targeted drug combination strategies. While lesions in the canonical MAPK signaling pathway occur in only 3-5% of newly diagnosed neuroblastoma specimens, it is now clear that neuroblastoma genomes evolve extensively under the selective pressure of intensely cytotoxic therapy. We have recently shown that 78% of relapsed neuroblastomas harbor mutations predicted to hyperactivate RAS signaling, many of which were clonally enriched after chemoradiotherapy ( Nat Genet, 2015 ) . The inhibition of MEK1/2 in xenograft models of MAPK-hyperactive neuroblastoma results in significant tumor growth delay, but progressive disease ultimately prevails. We hypothesized that a protein kinase siRNA screen performed in conjunction with trametinib treatment would identify synthetic lethal partners for MEK1/2 inhibition, and therefore, novel combination treatments to pursue in relapsed neuroblastoma patients characterized by MAPK-hyperactivating mutations. Methods: Neuroblastoma cell lines (N = 4) were optimized for cell plating, siRNA transfection, and trametinib treatment in 96-well format with the endpoint readout of cell viability (CellTiter-Glo). We chose a human protein kinase siRNA library (ON-TARGETplus, Dharmacon) to knockdown more than 700 kinases. Each cell line was screened in duplicate under both vehicle and trametinib treatments. Luminescence readings were log transformed and normalized (min-max) by plate according to positive (siPLK1) and negative (non-targeting siRNA) cell death controls. Hits were defined as genes that cooperated with trametinib in each cell line and in duplicate experiments. Results: Initial screening of two cell lines (NB-EBc1, SK-N-SH) yielded a list of 6 genes (COL4A3BP, MAPK3, RPS6KA6, SRMS, STK23, and TYR03) identified as hits. Two of these genes, MAPK3 (ERK1) and RPS6KA6 (RSK4), code for proteins downstream of MEK1/2, suggesting they may contribute to resistance to trametinib by bypassing MEK1/2 and activating MAPK effectors. Conclusions: Initial results of a synthetic lethal screen with trametinib suggest that dual inhibition of the MAPK kinase pathway via MEK1/2 and downstream effectors, such as ERK/RSK may provide synergistic inhibition of neuroblastoma. We are currently completing the screen in two additional neuroblastoma cell lines (SK-N-BE(2)-C and NB-16) and validating the initial findings. We expect to identify synthetic lethal partners for MEK1/2 inhibition, and therefore, trametinib-resistance pathways to exploit with novel combination therapies in neuroblastoma. Citation Format: Lori S. Hart, Pichai Raman, Grace Coggins, JulieAnn Rader, John M. Maris. The identification of synthetic lethal partners cooperating with MEK1/2 inhibition in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-126.

Abstract 4305: NMI as a biomarker of response to CDK4/6 inhibition in a preclinical model of neuroblastoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background Hyper-activation of the Cyclin D-CDK4/6 signaling pathway is a common feature of neuroblastoma. Previously, it has been shown that while a majority of preclinical models of neuroblastoma are highly sensitive to pharmacologic inhibition of CDK4/6 by LEE011 (Novartis Oncology), a subset are remarkably resistant. In an effort to aid in the selection of a patient population that will best benefit from this therapy, we sought to identify genomic biomarkers of response to CDK4/6 inhibition. Methods LEE011 IC50 values were determined using the RT-CES cell impedance assay, and gene expression was established at baseline using HuGene 1.0ST microarrays (Affymetrix). Expression data was then coupled with IC50 values to identify a set of genes significantly associated with LEE011 response via Pearson correlation. Using this gene set, a Lasso linear regression model was built in order to predict sensitivity to LEE011 based upon the expression of a subset of genes chosen to be most predictive of response. The model was tested in a separate cohort of neuroblastoma cell lines by comparing predicted responses to LEE011 with experimentally determined LEE011 IC50 values. Functional RNAi and over-expression studies are ongoing in order to validate genes identified to be correlated with or predictive of response. Results The Lasso model was able to predict the response of neuroblastoma cell lines to LEE011 based upon the expression pattern of 11 genes. A comparison of Lasso predictions to the LEE011 IC50 values of a separate panel of cell lines yielded a correlation coefficient of 0.63. The correlation of predicted to actual response across our entire cell line panel was 0.96, suggesting that response to CDK4/6 inhibition by LEE011 can be accurately predicted based on the expression of these genes. Of the 11 genes, NMI (N-myc and stat interactor) was prioritized for further validation and functional studies as it was identified by Pearson correlation to be the third most correlated gene with response (r = 0.86; p = 3.1×10-7) and because it interacts with MYCN, a known prognostic indicator in neuroblastoma. We next showed that NMI was expressed higher at both the mRNA and protein levels in neuroblastoma cell lines resistant (N = 6) to LEE011 compared to those demonstrating sensitivity to the compound (N = 16). Studies are ongoing to confirm the role of NMI as a predictor of resistance as well as to assess its contribution, if any, in desensitizing neuroblastoma to CDK4/6 inhibition. Conclusions We have identified NMI as a candidate biomarker of resistance to CDK4/6 inhibition in preclinical models of neuroblastoma. Further validation of this gene as a biomarker may ultimately help drive clinical decisions in selecting patients who would most benefit from CDK4/6 inhibition therapies, as well as in identifying novel drugs to combine with LEE011 for the treatment of neuroblastoma patients. Note: This abstract was not presented at the meeting. Citation Format: JulieAnn Rader, Pichai Raman, Lori Hart, Mike Russell, Kristina A. Cole, John M. Maris. NMI as a biomarker of response to CDK4/6 inhibition in a preclinical model of neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4305. doi:10.1158/1538-7445.AM2015-4305