Landscape analysis of adjacent gene rearrangements reveals BCL2L14-ETV6 gene fusions in more aggressive triple-negative breast cancer.

Triple-negative breast cancer (TNBC) accounts for 10 to 20% of breast cancer, with chemotherapy as its mainstay of treatment due to lack of well-defined targets, and recent genomic sequencing studies have revealed a paucity of TNBC-specific mutations. Recurrent gene fusions comprise a class of viable genetic targets in solid tumors; however, their role in breast cancer remains underappreciated due to the complexity of genomic rearrangements in this cancer. Our interrogation of the whole-genome sequencing data for 215 breast tumors catalogued 99 recurrent gene fusions, 57% of which are cryptic adjacent gene rearrangements (AGRs). The most frequent AGRs, BCL2L14-ETV6, TTC6-MIPOL1, ESR1-CCDC170, and AKAP8-BRD4, were preferentially found in the more aggressive forms of breast cancers that lack well-defined genetic targets. Among these, BCL2L14-ETV6 was exclusively detected in TNBC, and interrogation of four independent patient cohorts detected BCL2L14-ETV6 in 4.4 to 12.2% of TNBC tumors. Interestingly, these fusion-positive tumors exhibit more aggressive histopathological features, such as gross necrosis and high tumor grade. Amid TNBC subtypes, BCL2L14-ETV6 is most frequently detected in the mesenchymal entity, accounting for ∼19% of these tumors. Ectopic expression of BCL2L14-ETV6 fusions induce distinct expression changes from wild-type ETV6 and enhance cell motility and invasiveness of TNBC and benign breast epithelial cells. Furthermore, BCL2L14-ETV6 fusions prime partial epithelial-mesenchymal transition and endow resistance to paclitaxel treatment. Together, these data reveal AGRs as a class of underexplored genetic aberrations that could be pathological in breast cancer, and identify BCL2L14-ETV6 as a recurrent gene fusion in more aggressive form of TNBC tumors.

Fusion-associated carcinomas of the breast: Diagnostic, prognostic, and therapeutic significance.

Recurrent gene fusions comprise a class of viable genetic targets in solid tumors that have culminated several recent breakthrough cancer therapies. Their role in breast cancer, however, remains largely underappreciated due to the complexity of genomic rearrangements in breast malignancy. Just recently, we and others have identified several recurrent gene fusions in breast cancer with important clinical and biological implications. Examples of the most significant recurrent gene fusions to date include (1) ESR1::CCDC170 gene fusions in luminal B and endocrine-resistant breast cancer that exert oncogenic function via modulating the HER2/HER3/SRC Proto-Oncogene (SRC) complex, (2) ESR1 exon 6 fusions in metastatic disease that drive estrogen-independent estrogen-receptor transcriptional activity, (3) BCL2L14::ETV6 fusions in a more aggressive form of the triple-negative subtype that prime epithelial-mesenchymal transition and endow paclitaxel resistance, (4) the ETV6::NTRK3 fusion in secretory breast carcinoma that constitutively activates NTRK3 kinase, (5) the oncogenic MYB-NFIB fusion as a genetic driver underpinning adenoid cystic carcinomas of the breast that activates MYB Proto-Oncogene (MYB) pathway, and (6) the NOTCH/microtubule-associated serine-threonine (MAST) kinase gene fusions that activate NOTCH and MAST signaling. Importantly, these fusions are enriched in more aggressive and lethal breast cancer presentations and appear to confer therapeutic resistance. Thus, these gene fusions could be utilized as genetic biomarkers to identify patients who require more intensive treatment and surveillance. In addition, kinase fusions are currently being evaluated in breast cancer clinical trials and ongoing mechanistic investigation is exposing therapeutic vulnerabilities in patients with fusion-positive disease.

Low HIP1R mRNA and protein expression are associated with worse survival in diffuse large B-cell lymphoma patients treated with R-CHOP.

Huntingtin-interacting protein 1-related (HIP1R) is an endocytic protein involved in receptor trafficking, including regulating cell surface expression of receptor tyrosine kinases. We have previously shown that low HIP1R protein expression was associated with poorer survival in diffuse large B-cell lymphoma (DLBCL) patients from Denmark treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). In this multicenter study, we extend these findings and validate the prognostic and subtyping utility of HIP1R expression at both transcript and protein level. Using data mining on three independent transcriptomic datasets of DLBCL, HIP1R transcript was preferentially expressed in germinal center B-cell (GCB)-like DLBCL subtype (P<0.01 in all three datasets), and lower expression was correlated with worse overall survival (OS; P<0.01) and progression-free survival (PFS; P<0.05) in a microarray-profiled DLBCL dataset. At the protein level examined by immunohistochemistry, HIP1R expression at 30% cut-off was associated with GCB-DLBCL molecular subtype (P=0.0004; n=42), and predictive of OS (P=0.0006) and PFS (P=0.0230) in de novo DLBCL patients treated with R-CHOP (n=73). Cases with high FOXP1 and low HIP1R expression frequency (FOXP1(hi)/HIP1R(lo) phenotype) exhibited poorer OS (P=0.0038) and PFS (P=0.0134). Multivariate analysis showed that HIP1R<30% or FOXP1(hi)/HIP1R(lo) subgroup of patients exhibited inferior OS and PFS (P<0.05) independently of the International Prognostic Index. We conclude that HIP1R expression is strongly indicative of survival when utilized on its own or in combination with FOXP1, and the molecule is potentially applicable for subtyping of DLBCL cases.

A Novel Neoplastic Fusion Transcript, RAD51AP1-DYRK4, Confers Sensitivity to the MEK Inhibitor Trametinib in Aggressive Breast Cancers.

PURPOSE: Luminal B breast tumors are more aggressive estrogen receptor-positive (ER+) breast cancers characterized by aggressive clinical behavior and a high risk of metastatic dissemination. The underlying pathologic molecular events remain poorly understood with a paucity of actionable genetic drivers, which hinders the development of new treatment strategies. EXPERIMENTAL DESIGN: We performed large-scale RNA sequencing analysis to identify chimerical transcripts preferentially expressed in luminal B breast cancer. The lead candidate was validated by reverse transcription PCR in breast cancer tissues. The effects of inducible ectopic expression or genetic silencing were assessed by phenotypic assays such as MTS, transwell, and transendothelial migration assays, and by clonogenic assays to assess MEK inhibitor sensitivity. Subcellular fractionation, Western blots, and immunoprecipitation were performed to characterize the protein products and elucidate the engaged mechanisms. RESULTS: Here we report a novel tumor-specific chimeric transcript RAD51AP1-DYRK4 preferentially expressed in luminal B tumors. Analysis of 200 ER+ breast tumors detected RAD51AP1-DYRK4 overexpression in 19 tumors (9.5%), which is markedly enriched in the luminal B tumors (17.5%). Ectopic expression of RAD51AP1-DYRK4, but not wild-type RAD51AP1, leads to marked activation of MEK/ERK signaling, and endows increased cell motility and transendothelial migration. More importantly, RAD51AP1-DYRK4 appears to endow increased sensitivity to the MEK inhibitor trametinib through attenuating compensatory activation of HER2/PI3K/AKT under MEK inhibition. CONCLUSIONS: This discovery sheds light on a new area of molecular pathobiology of luminal B tumors and implies potential new therapeutic opportunities for more aggressive breast tumors overexpressing this fusion.

Therapeutic Targeting of Nemo-like Kinase in Primary and Acquired Endocrine-resistant Breast Cancer.

PURPOSE: Endocrine resistance remains a major clinical challenge in estrogen receptor (ER)-positive breast cancer. Despite the encouraging results from clinical trials for the drugs targeting known survival signaling, relapse is still inevitable. There is an unmet need to discover new drug targets in the unknown escape pathways. Here, we report Nemo-like kinase (NLK) as a new actionable kinase target that endows previously uncharacterized survival signaling in endocrine-resistant breast cancer. EXPERIMENTAL DESIGN: The effects of NLK inhibition on the viability of endocrine-resistant breast cancer cell lines were examined by MTS assay. The effect of VX-702 on NLK activity was verified by kinase assay. The modulation of ER and its coactivator, SRC-3, by NLK was examined by immunoprecipitation, kinase assay, luciferase assay, and RNA sequencing. The therapeutic effects of VX-702 and everolimus were tested on cell line- and patient-derived xenograft (PDX) tumor models. RESULTS: NLK overexpression endows reduced endocrine responsiveness and is associated with worse outcome of patients treated with tamoxifen. Mechanistically, NLK may function, at least in part, via enhancing the phosphorylation of ERα and its key coactivator, SRC-3, to modulate ERα transcriptional activity. Through interrogation of a kinase profiling database, we uncovered and verified a highly selective dual p38/NLK inhibitor, VX-702. Coadministration of VX-702 with the mTOR inhibitor, everolimus, demonstrated a significant therapeutic effect in cell line-derived xenograft and PDX tumor models of acquired or de novo endocrine resistance. CONCLUSIONS: Together, this study reveals the potential of therapeutic modulation of NLK for the management of the endocrine-resistant breast cancers with active NLK signaling.

DNMT1 is associated with cell cycle and DNA replication gene sets in diffuse large B-cell lymphoma.

Dysregulation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) is associated with the pathogenesis of various types of cancer. It has been previously shown that DNMT1 is frequently expressed in diffuse large B-cell lymphoma (DLBCL), however its functions remain to be elucidated in the disease. In this study, we gene expression profiled (GEP) shRNA targeting DNMT1(shDNMT1)-treated germinal center B-cell-like DLBCL (GCB-DLBCL)-derived cell line (i.e. HT) compared with non-silencing shRNA (control shRNA)-treated HT cells. Independent gene set enrichment analysis (GSEA) performed using GEPs of shRNA-treated HT cells and primary GCB-DLBCL cases derived from two publicly-available datasets (i.e. GSE10846 and GSE31312) produced three separate lists of enriched gene sets for each gene sets collection from Molecular Signatures Database (MSigDB). Subsequent Venn analysis identified 268, 145 and six consensus gene sets from analyzing gene sets in C2 collection (curated gene sets), C5 sub-collection [gene sets from gene ontology (GO) biological process ontology] and Hallmark collection, respectively to be enriched in positive correlation with DNMT1 expression profiles in shRNA-treated HT cells, GSE10846 and GSE31312 datasets [false discovery rate (FDR) <0.05]. Cell cycle progression and DNA replication were among the significantly enriched biological processes (FDR <0.05). Expression of genes involved in the activation of cell cycle and DNA replication (e.g. CDK1, CCNA2, E2F2, PCNA, RFC5 and POLD3) were highly correlated (r>0.8) with DNMT1 expression and significantly downregulated (log fold-change <-1.35; p<0.05) following DNMT1 silencing in HT cells. These results suggest the involvement of DNMT1 in the activation of cell cycle and DNA replication in DLBCL cells.

Cancer/testis antigen SPATA19 is frequently expressed in benign prostatic hyperplasia and prostate cancer.

Spermatogenesis-associated 19 (SPATA19) is a cancer/testis antigen overexpressed in various cancers. However, its protein expression profile in malignant or non-malignant tissues remains unknown. Thus, in this study, we investigated SPATA19 protein expression patterns in a panel of non-malignant human samples and primary prostate cancer (PCa) with or without benign prostatic hyperplasia (BPH) tissues. SPATA19 was absent in all non-malignant tissues investigated (n=14) except testis and prostate tissues. In terms of malignancies, all PCa cases were positive for SPATA19 exhibiting frequency between 20 and 100% (median 85%) with 63 (52.5%) and 57 (47.5%) cases demonstrating weak/moderate and strong intensities, respectively. Thirty-nine PCa cases (32.5%) contained BPH, and all BPH glands were SPATA19 positive (frequency between 20 and 100%; median 90%) with 13 (33.3%) demonstrating strong SPATA19 expression. Higher SPATA19 expression (higher frequency, intensity, or H-score) was not associated with overall survival or disease-specific survival (DFS) in all PCa cases. However, biochemical recurrence (BR) was associated with worse DFS (p = 0.005) in this cohort of 120 patients, and cases with strong SPATA19 intensity were associated with BR (p = 0.020). In conclusion, we showed that SPATA19 protein was frequently expressed in both BPH and PCa glands, and this warrants future investigations on its pathogenic roles in the disease.

DNMT1 is predictive of survival and associated with Ki-67 expression in R-CHOP-treated diffuse large B-cell lymphomas.

DNMT1 is a target of approved anti-cancer drugs including decitabine. However, the prognostic value of DNMT1 protein expression in R-CHOP-treated diffuse large B-cell lymphomas (DLBCLs) remains unexplored. Here we showed that DNMT1 was expressed in the majority of DLBCL cases (n = 209/230, 90.9%) with higher expression in germinal centre B-cell-like (GCB)-DLBCL subtype. Low and negative DNMT1 expression (20% cut-off, n = 33/230, 14.3%) was predictive of worse overall survival (OS; p < 0.001) and progression-free survival (PFS; p < 0.001). Nonetheless, of the 209 DNMT1 positive patients, 33% and 42% did not achieve 5-year OS and PFS, respectively, indicating that DNMT1 positive patients showed considerably heterogeneous outcomes. Moreover, DNMT1 was frequently expressed in mitotic cells and significantly correlated with Ki-67 or BCL6 expression (r = 0.60 or 0.44, respectively; p < 0.001). We demonstrate that DNMT1 is predictive of DLBCL patients' survival, and suggest that DNMT1 could be a DLBCL therapeutic target due to its significant association with Ki-67.