Oncogenic mechanisms and molecular targets in lymphoma

Staudt, Louis

Abstract

One method to discover new therapeutic targets in cancer is array-based comparative genomic hybridization coupled with gene expression profiling. Previously, the Staudt laboratory used gene expression profiling to define molecular subtypes of diffuse large B cell lymphoma (DLBCL), termed germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL and primary mediastinal B cell lymphoma (PMBL). The Staudt laboratory determined regions of copy number gain, amplification and deletion using array-based comparative genomic hybridization in 203 DLBCL biopsy samples. Using this approach, the Staudt laboratory uncovered a host of genetic aberrations that distinguished the subtypes from one another, providing compelling genetic evidence that these DLBCL subtypes should be considered as distinct disease entities. Gene expression profiling on the same samples was used to identify which genes in the affected genomic regions were altered in expression by the genomic aberrations. A region on chromosome 19 including the SPIB locus was gained or amplified in one quarter of ABC DLBCLs but not in GCB DLBCLs. SPIB was one of the most upregulated genes in cases with this genomic aberration. Moreover, laboratory demonstrated that the SPIB gene is juxtaposed to the immunoglobulin heavy chain locus by a chromosomal translocation in an ABC DLBCL cell line, leading to pronounced overexpression of SPIB. SPIB is a lymphoid-restricted ETS family transcription factor that is required for optimal B cell activation and germinal center responses. RNAi-mediated knock down of SPIB was lethal to all ABC DLBCL cell lines, establishing SPIB as a new oncogene and therapeutic target in this disease. The Staudt laboratory has embarked on a new initiative to discover oncogenic somatic mutations in lymphoid malignancies by cancer gene resequencing. Previously, in an RNA interference-based genetic screen, the Staudt laboratory discovered that a pathway involving CARD11, BCL10 and MALT1 was responsible for the constitutive NF-kB signaling in ABC DLBCL. In a clear validation of the RNAi-based genetic screen, the laboratory discovered recurrent somatic mutations in the CARD11 gene in ABC DLBCL tumor biopsies. All of the CARD11 mutations in DLBCL changed amino acids in one small domain that is predicted to adopt a coiled-coil structure. These CARD11 mutations created protein isoforms that constitutively engaged NF-kB signaling, apparently due to their ability to spontaneously form large cytosolic aggregates that colocalize with signaling proteins in the NF-kB pathway. Interference with the CARD11 coiled-coil domain was lethal to ABC DLBCL cells, thereby suggesting a method to attack CARD11 therapeutically. Among the downstream targets of NF-kB signaling in ABC DLBCL are the cytokines IL-6 and IL-10. Both cytokines signal through cell surface receptors linked to JAK family kinases, which phosphorylate and activate the transcription factor STAT3. STAT3 transactivates genes directly and also indirectly by interacting with the NF-kB transcription factors. Using RNA interference to knock down STAT3 expression in an ABC DLBCL cell line, the laboratory developed a gene expression signature of STAT3 activity. This STAT3 signature was present in a subset of primary ABC DLBCL tumors but absent in GCB DLBCLs. The subset of ABC DLBCL tumors with high STAT3 signature expression also expressed IL-6 and/or IL-10 and had nuclear phosphorylated STAT3. This ABC DLBCL subset also had high expression of a gene expression signature of NF-kB activation. From a therapeutic standpoint, ABC DLBCL cell lines were killed synergistically by a combination of a small molecule inhibitor of JAK family kinases with an IkB kinase beta inhibitor, which blocks NF-kB signaling.