Our laboratory has embarked on a new initiative to discover oncogenic somatic mutations in lymphoid malignancies by cancer gene resequencing. Previously, we discovered that a pathway involving CARD11, BCL10 and MALT1 (the CBM complex) was responsible for the constitutive NF-kB signaling in ABC DLBCL. We then identified recurrent somatic mutations in the CARD11 gene in 10% of ABC DLBCL biopsies that constitutively engaged NF-kB signaling. Subsequently, we defined 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 that affect their critical ITAM signaling motifs, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. These mutations do not initiate BCR signaling but rather potentiate ongoing BCR signaling. We discovered that ABC DLBCL BCRs recognize auto-antigens that initiate BCR signaling, and this antigenic engagement of the BCR was required for the survival of ABC DLBCL cell lines. We have recently investigated the role of ubiquitination in oncogenic signaling in ABC DLBCL. We discovered an important role for the linear ubiquitin chain assembly complex (LUBAC) in the survival of ABC DLBCL. This ubiquitin ligase associates withe CBM complex and is required for NF-kB engagement. We identified a rare germ line polymorphism in the coding region of the LUBAC subunit RNF31 that is enriched in ABC DLBCL. This creates a mutant isoform that promotes LUBAC assembly and NF-kB activation. We designed a peptide inhibitor based on this mutant that specifically kills ABC DLBCL cells, suggesting that LUBAC is a promising therapeutic target in this lymphoma subtype. Most recently, we demonstrated that the recruitment of LUBAC to the CBM complex depends on the action of two ubiquitin ligases, c-IAP1 and c-IAP2, These enzymes attache K63-linked polyubiquitin chains on BCL10 and themselves, thereby recruiting LUBAC as well as IkB kinase to the CBM complex by virtue of their ubiquitin binding domains. We also identified oncogenic signaling by the adapter protein MYD88 as the genetic basis for the JAK-STAT3 activation in ABC DLBCL. ABC DLBCLs depend on MYD88 and its associated kinases IRAK1 and IRAK4. By resequencing we identified MYD88 mutations in 39% of ABC DLBCLs, with 29% 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. 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 (see below). Lenalidomide has had activity in early phase clinical trials against ABC DLBCL, prompting us to investigate its mode of action in this setting. We discovered that lenalidomide induces the secretion of interferon beta by the ABC DLBCL cells, which 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, a transcription factor that plays an 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. Combined treatment with lenalidomide and ibrutinib virtually eliminated IRF4 expression, leading to synergistic killing of ABC DLBCLs. We have developed new methods to identify drug synergies in cancer together with Craig Thomas and colleagues in NCATS. Using a combinatorial high-throughput drug screening platform, we can evaluate the effects of two drugs in combination, each tested at 10 different concentrations, thereby identify concentration ranges in which the drugs synergize in killing cancer cells. As a test case, we used this platform to demonstrate that ibrutinib synergizes with inhibitors of the PI(3) kinase pathway, BCL2 inhibitors, and standard cytotoxic chemotherapeutic agents. Using this platform, we observed that a small molecule inhibitor of BET-domain chromatin proteins, JQ-1, synergized with ibrutinib and other inhibitors of BCR signaling in killing ABC DLBCL cells. This unexpected finding was complemented by functional studies showing that JQ-1, which acts in the nucleus, potently inhibits IkB kinase, the key regulator of the NF-kB pathway, which resides in the cytoplasm. This suggests that constitutive NF-kB activity in ABC DLBCL may be mediated, in part, by signals emanating in the nucleus. BET protein inhibitors emerge as new drug class that should be explored for the treatment of ABC DLBCL. Our most recent efforts to define the molecular basis of oncogenic signaling in lymphoma have employed quantitative proteomics (SILAC) together with new technologies to detect protein-protein interactions (proximity ligation assay and BioID2 biotinylase-tagged proteins). Using these methods in concert, we defined a multiprotein complex that we term the My-T-BCR supercomplex. We have shown that the My-T-BCR is responsible for the oncogenic NF-kB signaling in a variety of lymphoid malignancies. By CRISPR screening, TLR9 was required for the survival of ABC DLBCL cells. We used this knowledge to demonstrate, quite unexpectedly, that TLR9 associates with IgM in these lymphoma cells. Proximity ligation assays (PLAs) revealed that IgM and TLR9 associate with one another in an intracellular endolysosomal compartment. Remarkably, we found that the My-T-BCR complex contains the CBM adapter complex, which triggers NF-kB activation during BCR signaling, and MyD88, which triggers NF-kB activation during TLR signaling. We used PLA to show that active IkB kinase associates with this protein complex and that the complex is essential for all IKK-dependent NF-kB activation. These findings explained a puzzling observation in our clinical trial ibrutinib in DLBCL, namely that ABC tumors with mutations in the BCR subunit CD79B and also in MYD88 responded frequently. We showed that ibrutinib significantly decreases the number of My-T-BCR complexes in ABC DLBCL cells. Moreover, we used biopsy samples from the ibrutinib trial to show that the presence of the My-T-BCR complex correlated with response to ibrutinib. Thus, we traced the efficacy of ibrutinib in CD79B/MYD88 double mutant cells to its effect on the My-T-BCR supercomplex. Using SILAC quantitative proteomics, we made the further important observation that the My-T-BCR complex associates with the mTORC1 complex on lysosomal membranes and showed that mTORC1 inhibitors acted synergistically with ibrutinib to decrease My-T-BCR complex formation, suggesting that clinical trials of this combination could prove fruitful.
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