Elucidating oncogenic mechanisms in human B cell malignancies

This study consists of two pieces of work investigating haematological malignancies; Acute Lymphoblastic Leukaemia (ALL) and Diffuse Large B Cell Lymphoma (DLBCL). Firstly, Pre-B ALL represents the most common paediatric malignancy and despite increasingly improved outcomes for patients, ~ 20% of all patients diagnosed with ALL relapse. Activating mutations in the RAS pathway are common (~50%) and result in hyperactivation of the MAPK pathway. I identified Erk negative feedback control via DUSP6 to be crucial for NRASG12D-mediated pre-B cell transformation and investigated its potential as a therapeutic target. I showed that a small molecule inhibitor of DUSP6 (BCI) selectively induced cell death in patient-derived pre-B ALL cells; with a higher sensitivity observed in relapse pre-B ALL. I also discovered that a high level of Erk activity is required for proliferation of normal pre-B cells, but dispensable in leukemic pre-B ALL cells. In addition, I found that human B cell malignancies can be grouped into three categories that fundamentally differ in their ability to control Erk signalling strength. Secondly, DLBCL is the most common haematological malignancy and although potentially curable with chemotherapy, 40% of patients still succumb from their disease. Recent exome sequencing studies have identified hundreds of genetic alterations but, for most, their contribution to disease, or their importance as therapeutic targets, remains uncertain. I optimised a novel approach to screen the functional importance of these mutations. This was achieved by reconstituting non-malignant, primary, human germinal centre B cells (GC B cells) with combinations of wildtype and mutant genes to recapitulate the genetic events of DLBCL. When injected into immunodeficient mice, these oncogene-transduced GC B cells gave rise to tumours that closely resemble human DLBCL, reinforcing the biological relevance of this system. To screen potential tumour suppressor mutations in this system in a high throughput fashion, I developed a lymphoma-focused CRISPR library of 692 genes recurrently altered in B cell lymphomas. These experiments identified GNA13 as an unexpectedly potent tumour suppressor in human GC B cells and provided new understanding to its mechanism of action. These findings provide novel understanding of the complexity of oncogenic mechanisms in human B cell malignancies

Genetic manipulation and immortalized culture of ex vivo primary human germinal center B cells.

Next-generation sequencing has transformed our knowledge of the genetics of lymphoid malignancies. However, limited experimental systems are available to model the functional effects of these genetic changes and their implications for therapy. The majority of mature B-cell malignancies arise from the germinal center (GC) stage of B-cell differentiation. Here we describe a detailed protocol for the purification and ex vivo expansion of primary, nonmalignant human GC B cells. We present methodology for the high-efficiency transduction of these cells to enable combinatorial expression of putative oncogenes. We also describe alternative approaches for CRISPR-Cas9-mediated deletion of putative tumor suppressors. Mimicking genetic changes commonly found in lymphoid malignancies leads to immortalized growth in vitro, while engraftment into immunodeficient mice generates genetically customized, synthetic models of human lymphoma. The protocol is simple and inexpensive and can be implemented in any laboratory with access to standard cell culture and animal facilities. It can be easily scaled up to enable high-throughput screening and thus provides a versatile platform for the functional interrogation of lymphoma genomic data.D.H. was personally supported by a Clinician Scientist Fellowship from the Medical Research Council (MR/M008584/1). Research in the Hodson laboratory is supported by the Kay Kendall Leukaemia Fund, The Addenbrooke’s Charitable Trust and the Evelyn Trust. The Hodson laboratory receives core funding from Wellcome (203151/Z/16/Z) and MRC to the WellcomeMRC Cambridge Stem Cell Institute and from CRUK to the CRUK Cambridge Centre (A25117

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis

Genetic modification of primary human B cells to model high-grade lymphoma

Abstract: Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models

Genetic modification of primary human B cells to model high-grade lymphoma

Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models

Acquired CARD11 mutation promotes BCR independence in Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is an aggressive non-Hodgkin lymphoma that is molecularly and clinically heterogeneous. Gene expression studies have revealed how DLBCL can be divided into germinal center (GC) and activated B cell (ABC) subtypes. The ABC subtype is associated with constitutive activation of the NF-κB pathway, commonly as a consequence of genetic activation of the B cell receptor (BCR) pathway1. Components of the BCR pathway that are activated by mutation include CD79B, MYD88 and CARD11. Chronic stimulation of the BCR in ABC DLBCL may also result from engagement of the BCR by self antigens in the tumor microenvironment. These preclinical observations suggest a role for the targeted inhibitors of the BCR pathway in the treatment of DLBCL1.D.H. was supported by a Clinician Scientist Fellowship from the Medical Research Council (MR/M008584/1) and a project grant from the Kay Kendall Leukaemia Fund. The Hodson laboratory receives core funding from Wellcome and MRC to the Wellcome-MRC Cambridge Stem Cell Institute and from the CRUK Cambridge Cancer Centre

Functional interplay of Epstein-Barr virus oncoproteins in a mouse model of B cell lymphomagenesis.

Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis

Targeting MEK in vemurafenib-resistant hairy cell leukemia.

To the editor: Hairy cell leukemia (HCL) is a chronic, incurable B cell malignancy that presents with splenomegaly, bone marrow infiltration and cytopenias(1). Long remissions are typically achieved with purine analogues such as cladribine, but most cases will relapse and require further therapy. The discovery of the BRAF V600E mutation in almost all cases of HCL(2) has led to the widespread adoption of the BRAF inhibitor vemurafenib for treatment of patients relapsing after cladribine(3-5). Impressive responses are reported; however, relapse is inevitable(5, 6) and hematologists are now faced with a growing number of patients with vemurafenib-resistant HCL. The optimal management of these patients remains unclear.D.H. was personally supported by a Clinician Scientist Fellowship from the Medical Research Council (MR/M008584/1), G.C. by a Wellcome Trust Clinical PhD Fellowship (WT098051). W.Y. was supported by an International Collaboration Award from the Pathological Society of UK and Ireland. Research in M.D. lab was supported by grants from Bloodwise. Core support was received from the Cancer Research UK, Cambridge Cancer Centre. We thank Joanna Baxter and Cambridge Blood and Stem Cell Bank for sample collection and storage, and Calli Latimer and Claire Hardy for expert technical assistanc

Genetic modification of primary human B cells to model high-grade lymphoma

Abstract: Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models

Rlf–Mycl Gene Fusion Drives Tumorigenesis and Metastasis in a Mouse Model of Small Cell Lung Cancer

Abstract Small cell lung cancer (SCLC) has limited therapeutic options and an exceptionally poor prognosis. Understanding the oncogenic drivers of SCLC may help define novel therapeutic targets. Recurrent genomic rearrangements have been identified in SCLC, most notably an in-frame gene fusion between RLF and MYCL found in up to 7% of the predominant ASCL1-expressing subtype. To explore the role of this fusion in oncogenesis and tumor progression, we used CRISPR/Cas9 somatic editing to generate a Rlf–Mycl-driven mouse model of SCLC. RLF–MYCL fusion accelerated transformation and proliferation of murine SCLC and increased metastatic dissemination and the diversity of metastatic sites. Tumors from the RLF–MYCL genetically engineered mouse model displayed gene expression similarities with human RLF–MYCL SCLC. Together, our studies support RLF–MYCL as the first demonstrated fusion oncogenic driver in SCLC and provide a new preclinical mouse model for the study of this subtype of SCLC. Significance: The biological and therapeutic implications of gene fusions in SCLC, an aggressive metastatic lung cancer, are unknown. Our study investigates the functional significance of the in-frame RLF–MYCL gene fusion by developing a Rlf–Mycl-driven genetically engineered mouse model and defining the impact on tumor growth and metastasis. This article is highlighted in the In This Issue feature, p. 2945 </jats:sec