Our laboratory has embarked on a new initiative to discover oncogenic somatic mutations in lymphoid malignancies by cancer gene resequencing. Previously, in an RNAi-based genetic screen, we 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, our lab 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. Most recently, our Achilles heel screens allowed us to define a chronic active form of B cell receptor (BCR) signaling that activates NF-kB in ABC DLBCLs with wild-type CARD11. Such ABC DLBCLs die upon knockdown of BCR signaling components, including subunits of the B cell receptor itself. ABC DLBCLs have prominent clusters of the BCR in the plasma membrane, similar to antigen-stimulated normal B cells. Cancer gene resequencing revealed that over one fifth of ABC DLBCLs have mutations in the CD79B or CD79A subunits of the BCR. The most common mutations, present in 18% of ABC DLBCLs, involved a single tyrosine of the BCR signaling subunit, CD79B. These mutations affect the critical ITAM signaling motif, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. We have recently identified oncogenic signaling by the adapter protein MYD88 as the genetic basis for the JAK-STAT3 activation in ABC DLBCL. An RNAi screen in ABC DLBCL revealed the dependence of these cells on MYD88 and its downstream kinases IRAK1 and IRAK4. High-throughput RNA resequencing (RNA-seq) uncovered recurrent mutations in MYD88. MYD88 mutations are present in 39% of ABC DLBCLs, with 29% affecting changing one leucine in the MYD88 TIR domain to proline (L265P). The L265P mutant isoform spontaneously coordinates a signaling complexed involving IRAK1 and IRAK4, which turns on the NF-kB, JAK-STAT3 pathway, and type I interferon pathway. Small molecule inhibitors of IRAK4 kinase are selectively lethal to ABC DLBCL cells, offering new therapeutic prospects. ABC DLBCL cells are killed in a synergistic fashion by combined inhibition of the BCR and MYD88 pathways, suggesting the development of clinical trials combining drugs targeting both pathways. There are several new drugs entering early phase clinical trials that target the pathways we have implicated using our functional genomics methods. The B cell receptor signaling pathway affords many possible targets for the treatment of ABC DLBCL, notably BTK. We initiated a phase Ib clinical trial of a small molecule BTK inhibitor, termed ibrutinib, in patients with relapsed/refractory DLBCL, with gene expression profiling used for both diagnosis and pharmacodynamics. Thus far, ibrutinib monotherapy has induced many complete and partial responses in patients with ABC DLBCL, including those with primary refractory tumors that had never responded to any prior therapy. One patient has been in a sustained complete response for over 3 years, taking ibrutinib daily with no discernible side effects. Of note, ABC DLBCL tumors with and without CD79B mutations have responded, suggesting that BCR pathway addiction may be a prevalent feature in this lymphoma subtype. Based on these promising results, we have completed a multicenter phase 2 trial of ibrutinib in patients with relapsed/refractory DLBCL. Patients were assigned to the ABC or GCB subtypes by gene expression profiling and were given ibrutinib monotherapy. Ibrutinib produced a 41% response rate (complete + partial) in patients with relapsed/refractory ABC DLBCL, but only a 5% response rate in GCB DLBCL, as predicted by our laboratory investigations. Many of the responses have been durable, lasting from 6 months to over 1 year. Based on these promising results, ibrutinib is now being evaluated in newly diagnosed ABC DLBCL in combination with R-CHOP chemotherapy in a phase 3 randomized trial. We expect that ibrutinib will not be curative as a single agent in most patients with ABC DLBCL, leading to search for other agents that might synergize with ibrutinib in killing these lymphoma cells. Lenalidomide is a drug of relatively obscure action that has shown potent clinical activity in multiple myeloma and myelodysplastic syndromes. Lenalidomide has also had activity in early phase clinical trials against ABC DLBCL, prompting us to investigate its mode of action in this setting. Unexpectedly, we discovered that lenalidomide induces the secretion of interferon beta by the ABC DLBCL cells, and that this autocrine production is an important component of lenalidomide-induced cell death in ABC DLBCL. In addition, lenalidomide blocked B cell receptor signaling to NF-kB by decreasing expression of CARD11. Both of these phenotypes could be traced to the ability of lenalidomide to decrease expression of IRF4. By RNA interference and ChIP-seq analysis, we showed that IRF4 plays and essential survival role in ABC DLBCL. IRF4 is itself an NF-kB target gene, so that that agents that inhibit BCR signaling, such as ibrutinib, also decrease IRF4 expression. We found that combined treatment with lenalidomide and ibrutinib virtually eliminated IRF4 expression, leading to synergistic killing of ABC DLBCLs. These results support evaluation of this drug combination in clinical trials. Burkitt lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL (eBL) in developing countries, necessitating new strategies1. The normal germinal center B cell is the presumed cell of origin for both BL and diffuse large B cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may utilize different oncogenic pathways2. BL is subdivided into a sporadic subtype (sBL) that is diagnosed in developed countries, the EBV-associated endemic subtype (eBL), and an HIV-associated subtype (hivBL), but it is unclear whether these subtypes employ similar or divergent oncogenic mechanisms. Here we used high throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways in BL that cooperate with MYC, the defining oncogene of this cancer. In 70% of sBL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival PI(3) kinase pathway in BL, in part by augmenting tonic B cell receptor signaling. In 38% of sBL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL. In particular, current BL therapy entails high dose chemotherapy that cannot be delivered safely to elderly patients or to children in Africa with the endemic-form of BL. Potentially, combinations of targeted agents can be developed based on the molecular pathways that we have defined in BL that can be used safely and effectively in these settings.