Functional genomic screens for molecular targets in cancer

Division Of Basic Sciences - Nci

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Abstract

The Staudt laboratory has conducted RNA interference and CRISPR genetic screens for genes required for the proliferation and/or survival of human cell lines representing various subtypes of lymphoma and multiple myeloma. In diffuse large B cell lymphoma (DLBCL), previous work in the Staudt laboratory demonstrated that the anti-apoptotic NF-kB pathway is constitutively active in the activated B cell-like (ABC) subtypes of DLBCL but not the germinal center B cell-like (GCB) subtype of DLBCL, but the mechanisms underlying this abnormal signaling were enigmatic. The laboratory therefore conducted an RNAi screen in ABC and GCB DLBCL cell lines, searching for shRNAs that were selectively toxic for ABC DLBCL cells. This effort revealed that a signaling complex comprised of CARD11, MALT1, and BCL10 is required for the survival of ABC but not GCB DLBCL cell lines. In normal lymphocytes, this CARD11 complex engages the NF-kB pathway during antigen receptor signaling. The Staudt laboratory demonstrated that this signaling complex is responsible for the constitutive activation of the NF-kB pathway in ABC DLBCLs. Our RNA interference 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. Importantly, the BCR pathway offers a wealth of targets that can be exploited therapeutically, including several protein kinases (SRC-family kinases, SYK, BTK, PKCbeta) as well as PI(3) kinase. Ibrutinib, a clinically available kinase inhibitor of BTK kills ABC DLBCL cells by blocking their chronic active BCR signaling. Our RNA interference screens additionally revealed the MYD88 signaling pathway as essential for the survival of ABC DLBCL cells. MYD88 is a key adapter protein in the signaling pathway downstream of Toll-like receptors in innate immune cells. The RNAi screen identified shRNAs targeting MYD88 and its associated kinase IRAK1 as toxic for ABC DLBCL cells but not for cell line models of other lymphoma subtypes. This led us to discover recurrent mutations in MYD88 that create mutant isoforms that spontaneously activate the NF-kB pathway and are oncogenic. We investigated a recurrent amplicon in primary mediastinal large B cell lymphoma (PMBL) and Hodgkin lymphoma on chromosome 9p24 using RNAi screens. We uncovered three essential genes using an RNAi screen: JAK2, JMJD2C, and RANBP6. We showed that the kinase activity of JAK2 is activated in these lymphomas by autocrine IL-13 signaling. Surprisingly, JAK2 cooperated with JMJD2C in promoting survival of these lymphoma cells. RNA interference screening has proven highly successful in unraveling the key survival pathways in Burkitt lymphoma. We discovered that knockdown of the transcription factor TCF3 was lethal to Burkitt lymphoma cell lines, but not to cell line models of other aggressive lymphomas. By RNA resequencing, we found that TCF3 and its negative regulator are mutated in 70% of cases of sporadic Burkitt lymphoma, leading to TCF3 dependency. Our RNA interference screens also demonstrated that about two thirds of Burkitt lymphoma cell lines depend upon tonic signaling from the B cell receptor (BCR). Further, we discovered that TCF3 amplifies this tonic BCR signaling. Finally, we discovered that Burkitt lymphoma cell lines require cyclin D3/CDK6 for cell cycle progression. RNA resequencing revealed oncogenic activating mutations in cyclin D3 in 38% of cases. Surprisingly, pharmacologic inhibition of CDK6 causes apoptosis of Burkitt lymphoma cell lines and induced regression of established Burkitt lymphoma xenografts. An RNAi screen uncovered a crucial dependency of multiple myeloma cells on IRF4, a lymphoid-restricted transcriptional factor that is required for both lymphocyte activation and for plasmacytic differentiation. Of note, IRF4 is not translocated, amplified or mutated in most cases of multiple myeloma, and thus the dependency of myeloma cells on IRF4 exemplifies a new concept in cancer biology known as non-oncogene addiction. These results establish IRF4 as an important new therapeutic target in this lethal cancer. Indeed, we showed that the efficacy of the drug lenalidomide in myeloma is due in large measure to its ability to decrease IRF4 expression. In adult T cell leukemia/lymphoma, our RNAi screens uncovered a dependence on a transcriptional network controlled by BATF and IRF4 that is therapeutically targetable with BET protein inhibitors. Most recently, we have adopted the Cas9/CRISPR technology, which is an alternative way to inactivate genes. In this system, a short guide RNA (sgRNA) is used to direct the Cas9 endonuclease precise genomic locations. If the sgRNA targets the beginning of the coding region of a gene, the gene can be inactivated as a result of repeated cutting and deletion events. We have used bar-coded lentiviral libraries expressing sgRNAs to identify essential genes in lymphoid malignancies. We unexpectedly discovered that TLR9 is 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. Using quantitative proteomics we defined a MyD88-TLR9-BCR (My-T-BCR) supercomplex that coordinates all pro-survival NF-kB signaling in these ABC DLBCL cells. We have recently used "drug modifier" CRISPR screens to address the molecular pathways that cause resistance to drugs targeting oncogenic signaling in DLBCL. This effort has revealed new mechanisms of therapeutic resistance to BTK inhibitors in DLBCL. One mode of resistance is genetic inactivation of the tumor suppressor KLHL14, which we showed is an E3 ubiquitin ligase that increases the turnover of components of the B cell receptor in the endoplasmic reticulum. Another resistance mode relies on the epigenetic upregulation of RAC2, which can activate PLCG2 in lieu of BTK, thereby maintaining downstream NF-kB activity. In collaboration with Jon Yewdell, we used CRISPR screens to identify positive and negative regulators of MHC class I and class II expression in DLBCL, which uncovered novel mechanisms of immune evasion and identified mechanisms by which one could potentiate antigen presentation by DLBCL cells. 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.