Mike R. Russell

RNAi screen of the protein kinome identifies checkpoint kinase 1 (CHK1) as a therapeutic target in neuroblastoma

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC–Neuroblastoma-related (MYCN)–amplified (P < 0.0001) and high-risk (P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC50 values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC50 values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity (P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

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.

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.

Interleukin-1β promotes skeletal colonization and progression of metastatic prostate cancer cells with neuroendocrine features.

Despite the progress made in the early detection and treatment of prostate adenocarcinoma, the metastatic lesions from this tumor are incurable. We used genome-wide expression analysis of human prostate cancer cells with different metastatic behavior in animal models to reveal that bone-tropic phenotypes upregulate three genes encoding for the cytokine interleukin-1β (IL-1β), the chemokine CXCL6 (GCP-2), and the protease inhibitor elafin (PI3). The Oncomine database revealed that these three genes are significantly upregulated in human prostate cancer versus normal tissue and correlate with Gleason scores ≥7. This correlation was further validated for IL-1β by immunodetection in prostate tissue arrays. Our study also shows that the exogenous overexpression of IL-1β in nonmetastatic cancer cells promotes their growth into large skeletal lesions in mice, whereas its knockdown significantly impairs the bone progression of highly metastatic cells. In addition, IL-1β secreted by metastatic cells induced the overexpression of COX-2 (PTGS2) in human bone mesenchymal cells treated with conditioned media from bone metastatic prostate cancer cells. Finally, we inspected human tissue specimens from skeletal metastases and detected prostate cancer cells positive for both IL-1β and synaptophysin while concurrently lacking prostate-specific antigen (PSA, KLK3) expression. Collectively, these findings indicate that IL-1β supports the skeletal colonization and metastatic progression of prostate cancer cells with an acquired neuroendocrine phenotype.

CASC15-S Is a Tumor Suppressor lncRNA at the 6p22 Neuroblastoma Susceptibility Locus.

Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single-nucleotide polymorphism (SNP) associations reside within CASC15, a long noncoding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression.

The functional variant rs34330 of CDKN1B is associated with risk of neuroblastoma

The genetic aetiology of sporadic neuroblastoma is still largely unknown. We have identified diverse neuroblastoma susceptibility loci by genomewide association studies (GWASs); however, additional SNPs that likely contribute to neuroblastoma susceptibility prompted this investigation for identification of additional variants that are likely hidden among signals discarded by the multiple testing corrections used in the analysis of genomewide data. There is evidence suggesting the CDKN1B, coding for the cycle inhibitor p27Kip1, is involved in neuroblastoma. We thus assess whether genetic variants of CDKN1B are associated with neuroblastoma. We imputed all possible genotypes across CDKN1B locus on a discovery case series of 2101 neuroblastoma patients and 4202 genetically matched controls of European ancestry. The most significantly associated rs34330 was analysed in an independent Italian cohort of 311 cases and 709 controls. In vitro functional analysis was carried out in HEK293T and in neuroblastoma cell line SHEP‐2, both transfected with pGL3‐CDKN1B‐CC or pGL3‐CDKN1B‐TT constructs. We identified an association of the rs34330 T allele (‐79C/T) with the neuroblastoma risk (Pcombined = 0.002; OR = 1.17). The risk allele (T) of this single nucleotide polymorphism led to a lower transcription rate in cells transfected with a luciferase reporter driven by the polymorphic p27Kip1 promoter (P < 0.05). Three independent sets of neuroblastoma tumours carrying ‐79TT genotype showed a tendency towards lower CDKN1B mRNA levels. Our study shows that a functional variant, associated with a reduced CDKN1B gene transcription, influences neuroblastoma susceptibility.

Abstract 4758: Inhibition of checkpoint kinase 1 (Chk1) as a potential therapeutic for pediatric neuroblastoma

Neuroblastoma is a cancer derived from cells of the sympathetic nervous system that manifests with significant clinical heterogeneity. Although children diagnosed with low-risk neuroblastoma are very likely to be cured, high-risk patients are frequently resistant to even the most intensive of multi-modal regimens, and at least half of these children suffer relapse that is almost always fatal. To address the unmet need for novel therapeutic targets, our lab conducted an unbiased siRNA screen and determined that the loss of cell cycle checkpoint kinase 1 (Chk1) produced the most robust cytotoxic effect across multiple neuroblastoma cell lines. We subsequently confirmed that many neuroblastoma cell lines express constitutively phosphorylated Chk1 – a discovery that is supported by identical findings in tumors obtained from high-risk patients. Here we present evidence that the combination of a small molecule inhibitor of Chk1 with either irinotecan (SN-38) or cisplatin was synergistic or additive in vitro in cell lines sensitive to single agent Chk1 inhibition. Inhibition of Chk1 signaling as potential therapy in neuroblastoma is further supported by complimentary experiments in which we found that induction of shRNA specific for Chk1 results in marked cytotoxicity. To further understand the chemosensitizing aspect of Chk1 inhibition in neuroblastoma cells, we assayed the ATR/Chk1 pathway for potential biomarkers predictive of response. Candidate signaling partners in this pathway were isolated based upon either pharmacological inhibition or shRNA-induced silencing of Chk1, followed by hybridization to antibody microarrays. This technique has allowed for identification of Chk1-related signaling proteins involved in the DNA damage response pathway. Lastly, we have assessed the efficacy of Chk1 inhibition in an in vivo xenograft mouse model of pediatric neuroblastoma. We found that daily administration of a small molecule Chk1 inhibitor, currently undergoing Phase I clinical testing, was able to significantly impede the growth of NB1643 neuroblastoma xenografts (p=0.001). Efforts are currently underway in our laboratory to investigate a potentially synergistic effect in vivo through co-administration of this inhibitor with irinotecan or cisplatin. Taken together, these results provide a strong rationale for further studies to assess the use of Chk1 inhibition as either a single-agent or combinatorial therapy in pediatric neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4758. doi:10.1158/1538-7445.AM2011-4758

Abstract 5237: The long intergenic noncoding RNA LINC00340 is a neuroblastoma susceptibility gene

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Neuroblastoma is a clinically heterogeneous childhood tumor that presents as high-risk disease in 40% of diagnoses, and is fatal in roughly half of these patients despite aggressive multi-modal therapy. In an effort to understand the etiology of high-risk neuroblastoma, our lab previously undertook a genome-wide association study (2101 cases, 4202 controls), which identified a cluster of single-nucleotide polymorphisms on chromosome 6p that correlated with a significantly increased chance of developing of high-risk disease (p < 1e-15). Here we present evidence that these polymorphisms fall within a long intergenic non-coding RNA (LINC00340) termed cancer-associated susceptibility 15 gene (CASC15), which we hypothesize plays a critical role in the development of high-risk neuroblastoma through the dysregulation of neuronal growth and differentiation pathways. Methods: CASC15 expression from patient samples (n=251) was obtained via Affymetrix Human Exon 1.0ST arrays, whereas expression in cell lines was assessed by qRT-PCR. CASC15 isoform sequences were validated through the use of 5′ and 3′ RACE, and subcellular localization was visualized using RNA-FISH. CASC15 depletion was carried out using both siRNA and shRNA constructs, and cell viability and growth kinetics were measured using the CellTiter-Glo and Xcelligence platforms. Gene expression analyses of neuroblastoma cell lines stably silenced for CASC15 was done through hybridization to Human Transcriptome 2.0 arrays followed by gene set enrichment and Ingenuity pathway analysis. Results: Our data demonstrates that patients with high-risk neuroblastoma express significantly less CASC15 than do those with low-risk disease (p < 0.0001), and that CASC15 expression inversely correlates with overall survival (bonf p = 3.1e-05). Expression of CASC15 is variable amongst neuroblastoma cell lines (n= 21), and is predominantly nuclear. Depletion of CASC15 increases the rate of substrate-dependent adherent cellular growth, but has no effect on overall cell number or viability. In contrast, ectopic overexpression of CASC15 in neuroblastoma cell lines is not tolerated. Subsequent observation of cells with stable CASC15 depletion shows overt morphological changes suggestive of a less neuronal phenotype. Gene set expression and pathway analyses confirm that these cells undergo a significant downregulation of neuronal markers, while upregulating cell adhesion molecules. Conclusions: These data suggest that CASC15 may be integral for proper neuronal development, and that loss of CASC15 may leads to a less differentiated cell type, thereby contributing to the tumorigenesis of high-risk neuroblastoma. Citation Format: Mike R. Russell, Annalise Penikis, Derek Oldridge, Maura Diamond, Sharon Diskin, John Maris, Kristina Cole. The long intergenic noncoding RNA LINC00340 is a neuroblastoma susceptibility gene. [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 5237. doi:10.1158/1538-7445.AM2014-5237

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 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