Oncogenic mechanisms and molecular targets in lymphoma

Division Of Basic Sciences - Nci

Investigators

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Abstract

To discover oncogenic mechanisms and molecular targets in lymphoma, we have combined insights from structural and functional genomics. Our structural genomics effort integrates data from whole exome sequencing, array-based copy number analysis and RNA sequencing to identify recurrent genetic changes that alter oncogenic regulatory pathways. Our functional genomics effort involves whole genome CRISPR-Cas9 screening of cell line models of DLBCL genetic subtypes. 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. Various molecular mechanisms augment constitutive BCR signaling in lymphoma. For example, we identified KLHL14 as an E3 ligase that controls the level of BCR protein expression in ABC DLBCL cells. KLHL14 is to be a novel component of the ERAD pathway that is a quality control mechanism in the endoplasmic reticulum (ER). KLHL14 specifically regulates the half-life of BCR subunits in the ER. Of note, KLHL14 is recurrently inactivated by somatic mutations in the MCD genetic subtype of DLBCL, which is addicted to chronic active BCR signaling. Thus, KLHL14 mutations could serve as a biomarker for ABC DLBCL tumors that would respond to BTK inhibitors that block BCR-dependent NF-kB activation. In addition, ABC tumors genetically inactivate a host of negative regulators of BCR-dependent NF-kB signaling that foster addiction of these tumors to this signaling and responsiveness to pathway inhibitors 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. Despite frequent responses of ABC DLBCL tumors to ibrutinib monotherapy, therapeutic resistance is the rule rather than the exception. To model this, we have developed ibrutinib resistant cell line pools. Ibrutinib resistance is not generally genetic in these systems, but rather a stable epigenetic state that is, in part, dependent on the transcription factor TCF4. Paradoxically, ibrutinib-resistant cells have an increased dependence on BCR signaling and NF-?B, despite inactivation of BTK. The solution to this conundrum is that ibrutinib-resistant ABC cells upregulate the signaling adapter RAC2. RAC2 binds and activates PLCG2, a key mediator of NF-kB activation that is normally activated BTK phosphorylation. We have shown that RAC2 inhibitors are effective against ibrutinib-resistant ABC DLBCL and synergize with BCL2 inhibitors to overcome ibrutinib resistance. Most recently, we devised genome-wide CRISPR-Cas9 screens to identify regulators of IRF4, a direct transcriptional target of NF-kB and an indicator of proximal BCR signaling in ABC DLBCL. Unexpectedly, inactivation of N-linked protein glycosylation by the oligosaccharyltransferase-B (OST-B) complex reduced IRF4 expression. OST-B inhibition of BCR glycosylation reduced BCR clustering and internalization while promoting its association with CD22, which attenuated PI3 kinase and NF-kB activitation. By directly interfering with proximal BCR signaling, OST-B inactivation killed models of ABC and GCB DLBCL, supporting the development of selective OST-B inhibitors for the treatment of these aggressive cancers.