Contributions
Abstract: EP857
Type: E-Poster Presentation
Session title: Lymphoma Biology & Translational Research
Background
Non-Hodgkin’s lymphoma (NHL) is a diverse group of haematolymphoid malignancies, encompassing the more common diffuse large B-cell lymphoma (DLBCL) to the rarer T-cell lymphomas such as cutaneous T-cell lymphoma (CTCL). Several groups have performed comprehensive genomic analysis of various subtypes of NHL including DLBCL using whole exome sequencing (WES) and identified frequent occurrence of mutation in the DEAD box helicase 3, X-linked (DDX3X) gene. DDX3X is an RNA helicase and, depending on tumour type, plays a tumour suppressive or oncogenic role in cancer. However, its role in NHL remains unclear.
Aims
- To examine the mutational status of DDX3X in NHLs.
- To determine the molecular effects of DDX3X loss in cells derived from NHLs.
- To define functional involvements of DDX3X in NHLs.
Methods
We performed exome sequencing to identify genomic aberrations in DDX3X gene in DLBCL patients. Next, we generated CRISPR variants/ shRNA knockdown models of NHL cells line to mimic DDX3X mutation/loss. We determined the molecular effects of DDX3X loss by performing RNAseq analysis and immunoblot assay. We performed MTS and Annexin V/PI-based cell viability assays, BrdU proliferation assay, impedance-based metastasis assay and checkerboard assay to understand the functional involvement of DDX3X loss in NHLs.
Results
Targeted sequencing of DDX3X hotspots on exons 8-15 in a cohort of 158 unselected DLBCL subjects showed DDX3X mutations in 5 cases; whereas whole exome sequencing identified DDX3X mutations in 4 out of 9 patients with relapsed/refractory DLBCL treated with R-CHOP (rituximab + cyclophosphamide/doxorubicin/vincristine/ prednisone). DLBCL patients (n=223) with DDX3X mutations had worse 5-year overall survival (22%) compared to patients with wild-type DDX3X (72%, p=0.021). Using NHL cell lines, we showed that the expression of mutant DDX3X-R475C or DDX3X knockdown caused significant up-regulation of cyclin-D1 and increased phosphorylation of oncogenic STAT3, Akt and p42/44. We demonstrated that DDX3X loss enhances proliferation and metastasis in DDX3X R475C-mutant/depleted NHL cells. DDX3X loss increased resistance to doxorubicin and histone deacetylase targeting drugs in DLBCL and CTCL cells, respectively. Importantly, B- and T-cell lineage DDX3X-depleted cells remained sensitive to STAT3 inhibition.
Conclusion
In conclusion, our data suggest that loss-of-function mutations in DDX3X are important molecular determinants of disease aggressiveness and treatment response in NHL. These data will help improve risk stratification of NHL may identify new therapeutic options for a subgroup of patients with poor prognosis.
Keyword(s):
Abstract: EP857
Type: E-Poster Presentation
Session title: Lymphoma Biology & Translational Research
Background
Non-Hodgkin’s lymphoma (NHL) is a diverse group of haematolymphoid malignancies, encompassing the more common diffuse large B-cell lymphoma (DLBCL) to the rarer T-cell lymphomas such as cutaneous T-cell lymphoma (CTCL). Several groups have performed comprehensive genomic analysis of various subtypes of NHL including DLBCL using whole exome sequencing (WES) and identified frequent occurrence of mutation in the DEAD box helicase 3, X-linked (DDX3X) gene. DDX3X is an RNA helicase and, depending on tumour type, plays a tumour suppressive or oncogenic role in cancer. However, its role in NHL remains unclear.
Aims
- To examine the mutational status of DDX3X in NHLs.
- To determine the molecular effects of DDX3X loss in cells derived from NHLs.
- To define functional involvements of DDX3X in NHLs.
Methods
We performed exome sequencing to identify genomic aberrations in DDX3X gene in DLBCL patients. Next, we generated CRISPR variants/ shRNA knockdown models of NHL cells line to mimic DDX3X mutation/loss. We determined the molecular effects of DDX3X loss by performing RNAseq analysis and immunoblot assay. We performed MTS and Annexin V/PI-based cell viability assays, BrdU proliferation assay, impedance-based metastasis assay and checkerboard assay to understand the functional involvement of DDX3X loss in NHLs.
Results
Targeted sequencing of DDX3X hotspots on exons 8-15 in a cohort of 158 unselected DLBCL subjects showed DDX3X mutations in 5 cases; whereas whole exome sequencing identified DDX3X mutations in 4 out of 9 patients with relapsed/refractory DLBCL treated with R-CHOP (rituximab + cyclophosphamide/doxorubicin/vincristine/ prednisone). DLBCL patients (n=223) with DDX3X mutations had worse 5-year overall survival (22%) compared to patients with wild-type DDX3X (72%, p=0.021). Using NHL cell lines, we showed that the expression of mutant DDX3X-R475C or DDX3X knockdown caused significant up-regulation of cyclin-D1 and increased phosphorylation of oncogenic STAT3, Akt and p42/44. We demonstrated that DDX3X loss enhances proliferation and metastasis in DDX3X R475C-mutant/depleted NHL cells. DDX3X loss increased resistance to doxorubicin and histone deacetylase targeting drugs in DLBCL and CTCL cells, respectively. Importantly, B- and T-cell lineage DDX3X-depleted cells remained sensitive to STAT3 inhibition.
Conclusion
In conclusion, our data suggest that loss-of-function mutations in DDX3X are important molecular determinants of disease aggressiveness and treatment response in NHL. These data will help improve risk stratification of NHL may identify new therapeutic options for a subgroup of patients with poor prognosis.
Keyword(s):