Serial transcriptome analysis and cross-species integration identifies centromere-associated protein E as a novel neuroblastoma target

Abstract

Cancer genomic studies that rely on analysis of biopsies from primary tumors may not fully identify the molecular events associated with tumor progression. We hypothesized that characterizing the transcriptome during tumor progression in the TH-MYCN transgenic model would identify oncogenic drivers that would be targetable therapeutically. We quantified expression of 32,381 murine genes in nine hyperplastic ganglia harvested at three time points and four tumor cohorts of progressively larger size in mice homozygous for the TH-MYCN transgene. We found 93 genes that showed a linearly increasing or decreasing pattern of expression from the preneoplastic ganglia to end stage tumors. Cross-species integration identified 24 genes that were highly expressed in human MYCN-amplified neuroblastomas. The genes prioritized were not exclusively driven by increasing Myc transactivation or proliferative rate. We prioritized three targets [centromere-associated protein E (Cenpe), Gpr49, and inosine monophosphate dehydrogenase type II] with previously determined roles in cancer. Using siRNA knockdown in human neuroblastoma cell lines, we further prioritized CENPE due to inhibition of cellular proliferation. Targeting CENPE with the small molecular inhibitor GSK923295 showed inhibition of in vitro proliferation of 19 neuroblastoma cell lines (median IC(50), 41 nmol/L; range, 27-266 nmol/L) and delayed tumor growth in three xenograft models (P values ranged from P < 0.0001 to P = 0.018). We provide preclinical validation that serial transcriptome analysis of a transgenic mouse model followed by cross-species integration is a useful method to identify therapeutic targets and identify CENPE as a novel therapeutic candidate in neuroblastoma.

Figure 1. Harvested murine neuroblastomas represented clinically relevant stages of disease

Ultrasonography was used to monitor tumor growth. Tumors were harvested within four predetermined cohorts (n=6/cohort). Group A tumors were harvested once detected on ultrasound (median 0.08 ± 0.02 grams) and were macroscopically avascular with scant vascularity on CD34 staining (A). Group B tumors (0.35 ± 0.09 grams) displaced local structures, showed increased vascularity (B), but neither Group A or B tumors showed invasion of vasculature or metastases. Group C tumors (0.94 ± 0.22 grams) showed local invasion (C) but no metastasis, although 5/6 tumors had intratumoral invasion of vasculature. Group D (2.01g ± 0.59 grams) showed extensive local invasion (D) and intratumoral invasion of vasculature, with distant metastases in 3/6 animals. Group C and D tumors showed increasing vascularity that was grossly disorganized in the Group D tumors. CD34 immunohistochemistry images shown at 200×.

Figure 2

(A) Algorithm for identifying 24 candidates from ~32,000 transcripts. Transcripts showing a continuous increase or decrease in expression at clinically relevant stages of tumor progression, and in hyperplastic ganglia at day 0, 7 and 14 were identified using Spearman's method. Microscopic ganglia required an additional round of amplification so were analyzed separately. Of the 93 transcripts, cross-species integration was used to prioritize 24 biologically important genes that were overexpressed in human high-risk MYCN amplified neuroblastomas. (B) Heat map representation of 93 differentially expressed murine genes. Samples are grouped into ganglion samples (day 0, day 7, and day 14) and tumor samples (Groups A–D) along the X-axis. The 93 murine transcripts showing the most significant differential expression are shown along the Y-axis with red indicating greater expression, and green less expression. Four genes selected for functional evaluation are indicated. Note that murine Mycc and Mycn showed decreased expression with tumor progression. The full 93 gene set is described in Supplementary Table 2.

Figure 3. Prioritized transcripts showed increased expression with tumor progression but Myc transcriptional activity did not increase

Real-time PCR confirmed that three genes prioritized using a literature search (A) Cenpe, (B) Gpr49, (C) Impdh2 and a control gene not overexpressed in human MYCN amplified neuroblastoma (D) Tacstd1, showed a significant linear increase in expression with tumor size.

Figure 4. Gene expression signatures not driven by basal myc expression

(A) Murine neuroblastoma expression of human MYCN transgene mRNA (green) increased with tumor progression while murine Mycn (black) and Mycc (blue) expression decreased. Overall, the average MYCN, Mycn and Mycc expression did not increase with tumor progression (red). (B) Myc transcriptional activity was quantified using a clinically validated a priori defined gene expression signature (27) with a higher score indicating greater upregulation or downregulation of Myc targets. Myc transcriptional activity did not show a significant linear increase with tumor expression suggesting that alterations in MYCN transactivation did not exclusively drive differential gene expression.

Figure 5. siRNA knockdown prioritizes CENPE for further functional evaluation

We monitored proliferation of NB-1643, a MYCN-amplified human neuroblastoma cell line, using the RT-CES microelectronic cell sensor system. (A) The control gene, TACSTD1, which was not overexpressed in human MYCN amplified neuroblastomas showed minimal inhibition of proliferation with knockdown. Inhibition was modest for (B) GPR49 and (C) IMPDH2. (D) CENPE siRNA caused the greatest inhibition. The gene of interest is red and GAPDH (negative control) is blue. All experiments performed in triplicate, shown as mean ± SEM. PLK (positive control) showed 100% cell kill at 72 hours post transfection in all cases.

Figure 6. Pharmacological CENPE inhibition caused tumor growth delay in three human neuroblastoma xenografts

Mice were randomized to receive either GSK923295 125 mg/kg IP (red) or vehicle (blue: 96% acidified water, 2% DMAC, 2% CREM). Relative tumor volume (RTV), is the measured tumor volume divided by the enrolment tumor volume displayed as mean ± SEM. Linear mixed-effects analysis demonstrated smaller relative tumor volume in the treatment arm in all three xenografts. (A) NB-EBc1,p = 0.0001; (B) NB-1643, p = 0.018; and (C) NB-1691, p = 0.0018.