Genomic characterisation of Eμ-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene

The Eμ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eμ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eμ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.We acknowledge the following funding agencies: Leukaemia Foundation of Australia, Arrow Bone Marrow Transplant Foundation, National Health and Medical Research Council Australia, Cancer Council Victoria, Victorian Cancer Agency, Australian Cancer Research Foundation, Peter MacCallum Cancer Centre Foundation, National Institutes of Health

Identification of cooperating oncogenic lesions in Myc-driven lymphoma

© 2017 Dr. Marcus Patrick Henry LefebureMYC is a potent oncogene that is deregulated in nearly 50% of all human malignancies and as such, is considered an attractive molecular target for inhibition. However, MYC is rarely mutated, has no enzymatic activity that can be pharmacologically exploited and is expressed by normal cells leading to the current view that MYC is “undruggable” and indeed, its pharmacological inhibition has proved elusive. Therefore, discovering genes and pathways that interact with oncogenic MYC signalling and identifying them as potential therapeutic targets in cancers with ectopic MYC expression is of high clinical importance. The Eμ-Myc mouse has been utilised extensively as a faithful model of MYC-driven B cell lymphomagenesis. The Eμ-Myc transgene mimics the t(8;14) translocation apparent in Burkitt’s lymphoma, where ectopic Myc expression is driven by the Eμ- (IGH) promoter elements. Despite being driven by a single oncogene, Eμ-Myc lymphomas demonstrate remarkable heterogeneity indicating that the pathway to frank clonal neoplasia relies on oncogenic Myc signalling and the acquisition of at least one other mutation that cooperates with Myc. Hence, the Eμ-Myc model is a powerful tool in identifying MYC-cooperative genes and pathways. However, to date there has only been partial characterisation of the secondary, tertiary and quaternary mutations that can cooperate with Myc in driving Eμ-Myc lymphomagenesis. To identify somatically acquired Myc-cooperative lesions, massive-parallel sequencing was applied to spontaneous Eμ-Myc B cell lymphomas. Whole genome sequencing was used to map three copies of the Eμ-Myc transgene to chromosome 19 in the germline corresponding with an adjacent chromosome 19 segmental copy number gain. The chromosome 19 amplicon is in a region syntenic to an oncogenic region frequently amplified in human B cell malignancies. The chromosome 19 amplicon was demonstrated to undergo additional somatic gain in 50% of Eμ-Myc lymphoma. In addition to the identification of mutations in genes already implicated in Eμ-Myc lymphoma (Trp53, Cdkn2a, Nras, Kras), whole exome sequencing identified high frequency protein truncating mutations in Bcl6-co-repressor (Bcor). Furthermore, co-occurring tertiary driver lesions involving Cdkn2a (p19ARF) deletion and either Bcor or Ras mutations were identified in clonal Eμ-Myc lymphomas. RNAi and CRISPR-Cas9 mediated knockdown/knockout of Bcor in Eμ-Myc foetal liver hematopoietic stem cells reconstituted into recipient mice demonstrated significantly reduced latency of disease onset, validating Bcor as a tumor suppressor gene in the Eμ-Myc model. Gene-expression profiling of these Eμ-Myc tumours with forced Bcor-loss identified a reliable signature of Bcor loss that was distinct to Trp53 mutation signatures and was redolent of Tgfβ-pathway activation signature. The Eμ-Myc model of lymphoma has been heavily utilised but never fully genomically characterised until now. By applying next generation sequencing technology to a first generation animal model of cancer, this thesis challenges several persisting assumptions made about Eμ-Myc lymphoma. Firstly, data herein suggests that both oncogenic Myc expression along with the chromosome 19 amplification is the initiating driver event in Eμ-Myc lymphoma. This has obvious implications for the conclusions drawn in many publications predicated on the assumption that ectopic Myc expression is the exclusive initiating oncogenic lesion in Eμ-Myc lymphoma. Secondly, the discovery that homozygous deletion of Cdkn2a does not totally attenuate selective pressure for the acquisition of tertiary driver mutations indicates the significance of Cdkn2a deletion in Eμ-Myc lymphoma is overestimated. Thus, deductions made about the cooperative mechanism between CDKN2A and how it opposes proliferative MYC-signalling in human neoplastic transformation may need to be revisited. Finally, the identification of biologically functional high frequency Bcor mutations in Eμ-Myc lymphoma has defined a novel pathway that is potentially capable of restraining oncogenic MYC activity. That Bcor inactivation is reminiscent of Tgfβ-pathway enhancement is suggestive of perturbation of the oncogene induced senescence pathway. If this is the pathway through which Bcor exerts its tumour suppressive activity then it is feasible that dissection of this pathway will lead to the identification of novel therapeutic targets that can be selectively exploited in human malignancies in which MYC is oncogenically deregulated

Enduring undetectable MRD and updated outcomes in relapsed/refractory CLL after fixed-duration venetoclax-rituximab

The MURANO trial (A Study to Evaluate the Benefit of Venetoclax Plus Rituximab Compared With Bendamustine Plus Rituximab in Participants With Relapsed or Refractory Chronic Lymphocytic Leukemia [CLL]; ClinicalTrials.gov identifier #NCT02005471) reported superior progression-free survival (PFS) and overall survival (OS) with venetoclax-rituximab (VenR) vs bendamustine-rituximab (BR) in relapsed/refractory (R/R) CLL. Patients were randomized to 2 years of VenR (n = 194; rituximab for the first 6 months) or 6 months of BR (n = 195). Although undetectable minimal residual disease (uMRD) was achieved more often with VenR, the long-term implications of uMRD with this fixed-duration, chemotherapy-free regimen have not been explored. We report MRD kinetics and updated outcomes with 5 years' follow-up. Survival benefits with VenR vs BR were sustained (median PFS [95% confidence interval]: 53.6 [48.4, 57.0] vs 17.0 [15.5, 21.7] months, respectively, P <. 0001; 5-year OS [95% confidence interval]: 82.1% [76.4, 87.8] vs 62.2% [54.8, 69.6], P <. 0001). VenR was superior to BR, regardless of cytogenetic category. VenR-treated patients with uMRD at end of treatment (EOT; n = 83) had superior OS vs those with high-MRD+ (n = 12): 3-year post-EOT survival rates were 95.3% vs 72.9% (P =. 039). In those with uMRD at EOT, median time to MRD conversion was 19.4 months. Of 47 patients with documented MRD conversion, 19 developed progressive disease (PD); median time from conversion to PD was 25.2 months. A population-based logistic growth model indicated slower MRD median doubling time post-EOT with VenR (93 days) vs BR (53 days; P = 1.2 × 10−7). No new safety signals were identified. Sustained survival, uMRD benefits, and durable responses support 2-year fixed-duration VenR treatment in R/R CLL

Venetoclax Plus Rituximab in Relapsed Chronic Lymphocytic Leukemia: 4-Year Results and Evaluation of Impact of Genomic Complexity and Gene Mutations From the MURANO Phase III Study

PURPOSEIn previous analyses of the MURANO study, fixed-duration venetoclax plus rituximab (VenR) resulted in improved progression-free survival (PFS) compared with bendamustine plus rituximab (BR) in patients with relapsed or refractory chronic lymphocytic leukemia (CLL). At the 4-year follow-up, we report long-term outcomes, response to subsequent therapies, and the predictive value of molecular and genetic characteristics.PATIENTS AND METHODSPatients with CLL were randomly assigned to 2 years of venetoclax (VenR for the first six cycles) or six cycles of BR. PFS, overall survival (OS), peripheral-blood minimal residual disease (MRD) status, genomic complexity (GC), and gene mutations were assessed.RESULTSOf 389 patients, 194 were assigned to VenR and 195 to BR. Four-year PFS and OS rates were higher with VenR than BR, at 57.3% and 4.6% (hazard ratio [HR], 0.19; 95% CI, 0.14 to 0.25), and 85.3% and 66.8% (HR, 0.41; 95% CI, 0.26 to 0.65), respectively. Undetectable MRD (uMRD) at end of combination therapy (EOCT) was associated with superior PFS compared with low MRD positivity (HR, 0.50) and high MRD positivity (HR, 0.15). Patients in the VenR arm who received ibrutinib as their first therapy after progression (n = 12) had a reported response rate of 100% (10 of 10 evaluable patients); patients subsequently treated with a venetoclax-based regimen (n = 14) had a reported response rate of 55% (six of 11 evaluable patients). With VenR, the uMRD rate at end of treatment (EOT) was lower in patients with GC than in those without GC (P = .042); higher GC was associated with shorter PFS. Higher MRD positivity rates were seen with BIRC3 and BRAF mutations at EOCT and with TP53, NOTCH1, XPO1, and BRAF mutations at EOT.CONCLUSIONEfficacy benefits with fixed-duration VenR are sustained and particularly durable in patients who achieve uMRD. Salvage therapy with ibrutinib after VenR achieved high response rates. Genetic mutations and GC affected MRD rates and PFS