Search

Supplementary Figure 2 - PDF file 55K, Neuroblastoma tissue microarray. A tissue microarray demonstrates expression of RB in neuroblastoma that is higher in high-risk and MYCN amplified samples compared to low-risk or non-amplified samples (p = 0.03)

Supplementary Figure 5 - PDF file 138K, CDK4/6 inhibition does not induce cytotoxicity in neuroblastoma. (A) Caspase 3/7 activation and (B) PARP cleavage were not observed in cell lines with demonstrated sensitivity to LEE011. SN38, a known cytotoxic agent, was used as a positive control

Supplementary Table 1 - PDF file 78K, Genomic aberrations in the Cyclin D/CDK4/CDK6/RB pathway occur frequently in neuroblastoma cell lines

Supplementary Figure 4 - PDF file 88K, Protein level expression of FOXM1 in neuroblastoma

Supplementary Figure 1 - PDF file 56K, Expression of CDK4, CDK6, CCND1, and RB1 in neuroblastoma patients. Expression analysis of neuroblastoma primary tumors demonstrates that CDK4 expression, and to a lesser extent RB1 expression, is higher in high-risk neuroblastoma patients than in low risk patients

Supplementary Figure S1. Neuroblastoma cell lines are sensitive to binimetinib. Supplementary Figure S2. Gene expression profiling of human neuroblastoma cell lines with regards to relative sensitivity to MEK1/2 and CDK4/6 inhibition Supplementary Figure S3. Combined MEK and CDK4/6 inhibition does not induce significant levels of apoptosis or senescence. Supplementary Figure S4. G1 arrest induced by MEK1/2 and CDK4/6 inhibition is reversible. Supplementary Figure S5. Gene expression profiling of human neuroblastoma cell lines with regards to the synergy observed from combined binimetinib-ribociclib treatment. Figure S6. Neuroblastoma xenografts are sensitive to combined binimetinib-ribociclib treatment.

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.

Supplementary Figure 6 - PDF file 75K, Statistical analysis of in vivo growth suppression. (A) Growth rates of BE2C, 1643, and EBC1 xenografts treated with 200 mg/kg LEE011 or vehicle, as determined by linear mixed effects analysis. (B) Summary table of statistics

Table S1. Specific mutations of genes listed in Table 1. Table S2. Foundation Medicine sequencing calls of cancer-specific gene panel. Table S3. Gene lists corresponding to microarray analysis described in Figures 1A and 1B: (A) binimetinib and (B) ribociclib. Significant genes (in bold type) are defined as having an adjusted p-value of 0.25 or less.

This document contains Supplementary information, including materials and methods as well as Figure Legends for the Supplementary Figures.

Supplementary Table 2 - PDF file 48KB, Copy number variations within the Cyclin D/CDK4/CDK6/RB pathway occur in high-risk neuroblastoma patients

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.

Supplementary Figure 3 - PDF file 127K, MYCN expression is inversely correlated with LEE011 sensitivity. (A) High mRNA expression of MYCN correlates with low LEE011 IC50s (r = -0.55, p = 0.02). This correlation is further strengthened (r = -0.7, p = 0.003) by the removal of two outliers, SKNSH (IC50 = 148 nM) and its subclone, SY5Y (IC50 = 154 nM). (B) Sensitive cell lines also tend to express higher levels of MYCN protein than resistant cell lines

PDF file - 126K, Dual inhibition of Chk1 and Wee1 significantly impairs tumor growth in vivo. Mice were treated with either 30 mg/kg of single agent MK-1775 (targeting Wee1), single-agent MK-8776 (targeting Chk1) or simultaneous MK-1775 and MK-8776. Mixed linear effects model was used to calculate the rate of tumor growth in each condition, and demonstrated that dual inhibition was efficacious in both SKNAS and NB1643 xenograft models. Mice harboring NB1643 xenografts were treated for 48h, tumors were resected and target engagement was validated by western blot analysis

The factors regulating the bone tropism of disseminated prostate cancer cells are still vaguely defined. We report that prostate cancer cells that metastasize to the skeleton respond to human bone marrow with a robust stimulation of the phosphatidylinositol 3-kinase/Akt pathway, whereas prostate cells that lack bone-metastatic potential respond negligibly. The majority of this Akt activation is dependent on α-platelet–derived growth factor receptor (α-PDGFR) signaling, which was shown using the small-molecule inhibitor of PDGFR signaling AG1296. Low concentrations of PDGF-AA and PDGF-BB found in bone marrow aspirates, which were detected by ELISA, do not account for the high levels of α-PDGFR signaling. Additionally, neutralizing PDGF binding using a α-PDGFR–specific antibody (IMC-3G3) failed to produce a significant inhibition of bone marrow–induced Akt activation. However, the inhibitory effect of IMC-3G3 rivaled that of AG1296 when incubation was done under conditions that stimulated α-PDGFR internalization. We conclude that α-PDGFR is activated by multiple soluble factors contained within human bone marrow, in addition to its natural ligands, and this transactivation is dependent on receptor localization to the plasma membrane. Therefore, α-PDGFR expression may provide select prostate phenotypes with a growth advantage within the bone microenvironment. [Cancer Res 2007;67(2):555–62]

PDF file - 42K, Therapies targeting Chk1/Wee1 in combination with chemotherapy demonstrates synergy in most neuroblastoma cells. Ten neuroblastoma cell lines were tested for synergistic interactions between MK-1775 or MK-8776 in combination with either SN-38 (irinotecan) or gemcitabine. Combination indices were determined by increasing concentrations of both drug simultaneously (based on multiples of each drugs individual IC50), utilizing the Chou-Talalay method. The majority of neuroblastoma lines demonstrated synergy (denoted by a CI value < 0.7) in combination with chemotherapy, and particularly in the MK-1775 + MK-8776 combination group

Supplementary Data 2 from Human Bone Marrow Activates the Akt Pathway in Metastatic Prostate Cells through Transactivation of the α-Platelet–Derived Growth Factor Receptor

List of imputation results at the 6p22.3 region.

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. Cancer Res; 73(11); 3297–305. ©2013 AACR.

PDF file - 48K, Wee1 and Chk1 expression are elevated in high-risk, MYCN-amplified neuroblastoma. Primary tumors were obtained from 251 patients at diagnosis (221 high-risk, 30 low-risk; 68 MYCN amplified, 183 MYCN non-amplified), and were run on Affymetrix Human Exon 1.0 ST expression microarrays. Both Wee1 and Chk1 expression were expressed at a significantly higher rate in both the MYCN amplified and high-risk conditions

PDF file - 128K, Target engagement was validated for several cell lines via western blot analyses. Here, RPE-1, SK-N-AS, KELLY and NB-1691 cells are shown following treatment with increasing concentrations of MK-8776 and MK-1775 for 16h. Inhibition of Chk1 (via MK-8776) and Cdc2 phosphorylation (via MK-1775) was observed in sensitive cell lines

Supplementary Data 1 from Human Bone Marrow Activates the Akt Pathway in Metastatic Prostate Cells through Transactivation of the α-Platelet–Derived Growth Factor Receptor

rs6939340 genotype and expression of 6p22.3 gene products.

PDF file - 1996K, Supplementary Figure 1. Metastatic progression of different human prostate cancer cells in the skeleton of animal model. Supplementary Figure 2. Identification of pro-metastatic genes regulated by PDGFRa in PC3 cells. Supplementary Figure 3. Genes regulated by PDGFRa in DU-145 cells, which did not acquire bone-metastatic behavior upon exogenous expression of this receptor. Supplementary Figure 4. Two single-cell progenies of PC3-ML cells with high bone-metastatic potential. Supplementary Figure 5. Analysis of IL-1b expression and secretion from prostate cancer cells. Supplementary Figure 6. PDGFRa regulates Elafin and CXCL6 expression in prostate cancer cells. Supplementary Table 1. Contingency table of TMA data showing that prostate tumors with Gleason scores (≥7) expressed higher levels of the IL-1b protein as compared to tumors with lower Gleason scores (

Depletion of the long isoform of CASC15 does not impact neuroblastoma cell viability.

Full Table for the CASC15-S Cox Statistical Model.

Supplementary Methods

Evidence supporting multiple lncRNA transcript isoforms mapping to chromosome 6p22.3.

Additional expression data for CASC15 isoforms in neuroblastoma cell lines and patients.

PDF file - 133K, Expression and activity of Wee1 is prevalent in neuroblastoma. (A) A secondary panel of neuroblastoma cell lines demonstrated Wee1 expression and activation (evidenced by phospho-Ser642) by western blot. Cdc2 activity was observed in all neuroblastoma lines. (B) A variety of adult cancer lines were tested for Wee1 phosphorylation, and were generally much lower than that seen in neuroblastoma (far right lanes)

Supplementary Figure Legends

Bioinformatical support for CASC15-S as a long noncoding transcript.

Cell viability assays for neuroblastoma cell lines stably depleted of CASC15-S.

PDF file - 55K

Results of Sanger sequencing from 5' and 3'-RACE products of CASC15-S.

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.

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.

TPS249 Background: Human kallikrein-2 (hk2) is a member of the kallikrein family, is produced by prostatic epithelium, has 80% DNA sequence homology with PSA, is both circulating and bound to prostatic tissue, and is highly expressed even in high grade tumors. hK2 holds the promise of serving as a new therapeutic target for radiopharmaceuticals in prostate cancer. h11B6 is a humanized antibody directed to an epitope found on the catalytically active form of hK2, which is permanently internalized after binding. This is a first-in-human trial of [111In]-DOTA-h11B6 to determine the radio-immunotherapeutic potential of targeting hK2 with nanomolar affinity. The trial will determine the most favorable antibody mass as well as assess tumor targeting. Methods: To determine the optimal antibody mass, two cohorts of patients will be studied. All patients will receive 2 mg of [111In]-labeled h11B6. The first cohort consists of escalating amounts of antibody mass in 2-6 patients: 2, 10 and 20 mg total antibody respectively. Patients will undergo serial whole-body images for up to a week, along with assessments of serum clearance kinetics and normal organ residence time. The lowest mass amount of antibody that demonstrates favorable biodistribution and evidence of radioactivity accumulation in tumor will be used for the second cohort. Up to 6 patients will be studied in the second cohort, which will focus on assessment of antibody tumor targeting. Following this optimization and targeting imaging trial, a phase 1 dose-escalation radioimmunotherapy trial may be performed. Clinical trial information: NCT04116164.

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.

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. Cancer Res; 73(11); 3297–305. ©2013 AACR.

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 IC50 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 (IC50s 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. Cancer Res; 73(2); 776–84. ©2012 AACR.

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 IC 50 values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC 50 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.

TP53 is the most frequently mutated gene in human malignancies; however, de novo somatic mutations in childhood embryonal cancers such as neuroblastoma are rare. We report on the analysis of three independent case-control cohorts comprising 10290 individuals and demonstrate that rs78378222 and rs35850753, rare germline variants in linkage disequilibrium that map to the 3' untranslated region (UTR) of TP53 and 5' UTR of the Δ133 isoform of TP53, respectively, are robustly associated with neuroblastoma (rs35850753: odds ratio [OR] = 2.7, 95% confidence interval [CI] = 2.0 to 3.6, P combined = 3.43×10(-12); rs78378222: OR = 2.3, 95% CI = 1.8 to 2.9, P combined = 2.03×10(-11)). All statistical tests were two-sided. These findings add neuroblastoma to the complex repertoire of human cancers influenced by the rs78378222 hypomorphic allele, which impairs proper termination and polyadenylation of TP53 transcripts. Future studies using whole-genome sequencing data are likely to reveal additional rare variants with large effect sizes contributing to neuroblastoma tumorigenesis.

Background: Prostate cancer is the most common cancer and second most frequent cause of cancer-related deaths for men in the United States. AR-axis inhibitors have been shown to improve both progression-free survival (PFS) and overall survival (OS) in castration resistant prostate cancer (CRPC) patients however an estimated 50% of patients will go on to develop resistance to these therapies. Some mechanisms of resistance observed in the clinic include Androgen Receptor (AR) dependent events such as increased AR copy number, activating point mutations in AR, or splice variants (ARV7) both of the latter leading to constitutively active versions of AR. Alternatively, AR-independent resistance mechanisms include up-regulation of other oncogenic signaling pathways such as FGFR and PI3/Akt and induction of the nuclear hormone Glucocorticoid Receptor (GR). Finally, an increase in the emergence of a neuroendocrine phenotype is observed in the clinic with CRPC patients and is thought to be associated with chronic AR-antagonist treatment. To better understand the specific proteomic and genomic changes that occur during chronic AR inhibitor treatment we developed two enzalutamide (ENZA) resistant cell lines and performed targeted proteomic and global gene expression analysis. Methods: We generated LnCaP and VCAP ENZA-resistant (ENZA-Res) prostate cancer cell lines. We performed gene expression analysis by using both cDNA microarray and RNA-SEQ techniques to identify novel or differential gene expression patterns between ENZA-Res and sensitive phenotypes. We also employed a targeted proteomic approach to identify changes in the relative expression levels of proteins that have historically been associated with anti-androgen resistance including, ARV7, GR and SGK-1. Results: We have shown that ENZA-Res cell lines can undergo a significant change in morphology that is associated with an elongated neuronal-like appearance. This phenotype is associated with an induction in the mRNA expression of neuroendocrine markers, CHGA and REST along with a strong induction of GR mRNA and protein expression. We have shown that the neuroendocrine derived prostate cancer cell line, H660, also demonstrates high GR mRNA and protein content suggesting that GR may have a role in the maintenance of neuroendocrine and/ or ENZA-Res phenotypes when AR is absent. In VCaP ENZA-Res cells a significant increase in ARV7 mRNA and protein content was observed compared to parental ENZA sensitive VCaP cells. Lastly, we employed a combination of cDNA microarray and RNA-SEQ techniques to identify a molecular signature associated with anti-androgen resistance. We have identified a unique subset of genes that are differentially up-regulated in ENZA-Res cell lines compared to ENZA-sensitive cell lines. This novel molecular signature identifies several candidate genes as potential therapeutic targets that may be important in mediating anti-androgen resistance. Citation Format: Jennifer Hertzog, Mike Russell, Michael Quigley, Marco Gottardis, Theresa McDevitt. Genomic and proteomic characterization of anti-androgen resistant cell lines. [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 5040.

Purpose: Platelet-derived growth factor α (PDGFRα) is highly expressed in primary prostate cancer and associated skeletal metastases. Here, we tested whether targeting this receptor could impair metastatic colonization and progression, as well as prolong survival, either as primary or as combination therapy. Experimental Design: We used a preclinical animal model of metastasis in which PC3-ML human prostate cancer cells are inoculated directly in the blood circulation. First, the humanized, monoclonal antibody IMC-3G3 was administered to mice bearing established skeletal metastases. Second, we targeted the stromal PDGFRα with IMC-1E10, an antibody specific for the murine receptor. Third, IMC-3G3 and the bisphosphonate zoledronic acid (ZA), administered separately or in combination, were tested on the progression of skeletal lesions and overall survival. In addition, the ability of IMC-3G3 and ZA to impair initial colonization of the bone marrow by prostate cancer cells was investigated. Results: The blockade of PDGFRα on prostate cancer cells by IMC-3G3 reduces the size of established skeletal metastases, whereas the IMC-1E10 antibody directed against the stromal PDGFRα fails to inhibit metastatic progression. IMC-3G3 and ZA, either separately or in combination, significantly slow tumor growth and seem to prolong survival. Lastly, the blockade of PDGFRα by IMC-3G3 inhibits the initial phase of bone colonization, whereas ZA is ineffective at this stage. Conclusion: This study presents compelling evidence that targeting PDGFRα with IMC-3G3 delays the progression of early metastatic foci and reduces the size of more established lesions. In addition, IMC-3G3, either alone or in combination with ZA, prolongs survival in animal models. Clin Cancer Res; 16(20); 5002–10. ©2010 AACR.

Prostate adenocarcinoma is the second leading cause of cancer death among men, due primarily to the fact that the majority of prostate cancers will eventually spread to the skeleton. Metastatic dissemination requires a complex series of coordinated events that result in cells that escape from the primary tumor into the circulation and eventually colonize a distant organ. The ability of these cells to evolve into macroscopic metastases depends strongly on their compatibility with, and ability to utilize, this new microenvironment. We previously showed that bone-metastatic prostate cancer cells exposed to human bone marrow respond by activation of cell survival pathways, such as phosphoinositide 3-kinase/Akt, and that these events are mediated by the α-receptor for platelet-derived growth factor (PDGFRα). Our studies and others have shown that PDGFRα may be activated by mechanisms independent of PDGF ligand binding. Here, we provide conclusive evidence that soluble components of human bone marrow can activate PDGFRα through a mechanism that does not require the canonical binding of PDGF ligand(s) to the receptor. In particular, we found that dimerization of PDGFRα monomers is not induced by human bone marrow, but this does not prevent receptor phosphorylation and downstream signaling from occurring. To establish the relevance of this phenomenon in vivo, we used a PDGFRα mutant lacking the extracellular ligand-binding domain. Our studies show that this truncated PDGFRα is able to restore bone-metastatic potential of prostate cancer cells as effectively as the full-length form of the receptor. Cancer Res; 70(10); 4195–203. ©2010 AACR.

Bone resorption by osteoclasts is thought to promote the proliferation of prostate cancer cells disseminated to the skeleton (Mundy, 2002). Using a mouse model of experimental metastasis, we found that although latestage metastatic tumors were indeed surrounded by osteoclasts, these cells were spatially unrelated to the small foci of cancer cells in early-stage metastases. This is the first evidence that survival and growth of disseminated prostate cancer cells immediately after their extravasation may not depend on osteoclast involvement. Interestingly, prostate cancer cells expressing the a-receptor for platelet-derived growth factor (PDGFRa) progress during early-stages of skeletal dissemination, whereas cells expressing lower levels or lacking this receptor fail to survive after extravasation in the bone marrow. However, non-metastatic cells acquire bone-metastatic potential upon ectopic overexpression of PDGFRa. Finally, functional blockade of human PDGFRa on prostate cancer cells utilizing a novel humanized monoclonal antibody— soon to undergo phase-II clinical trials—significantly impairs the establishment of early skeletal metastases. In conclusion, our results strongly implicate PDGFRa in prostate cancer bone tropism through its promotion of survival and progression of early-metastatic foci, providing ground for therapeutic strategies aimed at preventing or containing the initial progression of skeletal metastases in patients affected by prostate adenocarcinoma. Oncogene (2009) 28, 412–421; doi:10.1038/onc.2008.390; published online 13 October 2008

Children with neuroblastoma exhibit marked variability in outcome based on age at diagnosis, disease stage and tumor biologic characteristics. Genomic amplification of MYCN is a potent oncogenic driver and negative prognostic marker in neuroblastoma patients. Therefore, there is a pressing need to identify therapeutic agents that target MYCN gene expression. The bromodomain and extra-terminal (BET) family of proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Previous reports demonstrate that BET inhibition (BETi) significantly attenuates cellular proliferation in numerous cancer models, perhaps via modulation of the MYC and/or MYCN oncogenes. Here, we show that potent BET inhibition induces anti-tumor effects in preclinical neuroblastoma models. Specifically, GSK726 and GSK762 (GlaxoSmithKline) were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. Neuroblastoma cell lines (n=22) were treated with GSK726 to calculate IC50 values in a luminescence-based cell viability assay and differential sensitivity was observed with an IC50 range of 27 nM to 9.5 µM. In sensitive cell lines, cell cycle distribution and induction of apoptosis were also measured. In these cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis as measured by cleaved-PARP. To assess in vivo efficacy, GSK762 was subcutaneously administered in xenograft models and in genetically engineered neuroblastoma mouse models overexpressing MYCN and MYCN/ALK F1174L in the neural crest. In both models, GSK762 treatment resulted in tumor growth delay. Further assessment of results seen in vitro and in vivo indicated that MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50 values by comparing sensitive (n=6; IC50940 nM) neuroblastoma cell lines. Gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor. Univariate analysis, with a false discovery rate less than 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) that were differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (p=7.92x10-7). In addition, high DACH1 expression was correlated with poor patient outcome (p=1.74x10-5). As a result, high DACH1 expression serves as a candidate biomarker for future studies with respect to sensitivity to BETi. Ultimately, these studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies. Citation Format: Jason R. Healy, Robert W. Schnepp, Lori S. Hart, Pichai Raman, Laura Danielson, Michael Russell, Priya Khurana, Maria Gagliardi, Ryan M. Kinsey, Anastasia Wyce, Olena Barbash, Peter J. Tummino, Louis Chesler, John M. Maris. Antitumor activity and sensitivity evaluation of novel BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B34.

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