Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell mediated immunosurveillance1,2. The success of therapies that disrupt PD-L1 mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression1. Using a genome-wide CRISPR/Cas9 screen we identified the uncharacterized protein CMTM6 to be a critical regulator of PD-L1 in a broad range of cancer cells. CMTM6 is a ubiquitously expressed, protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome we find that CMTM6 displays remarkable specificity for PD-L1. Importantly, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC Class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour specific T-cell activity in vitro and in vivo. These findings provide novel insights into the biology of PD-L1 regulation, identify a previously unrecognised master regulator of this critical immune checkpoint and highlight a new potential therapeutic target to overcome immune evasion by tumour cells.

SummaryLoss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

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Targeted therapies against disruptor of telomeric silencing 1-like (DOT1L) and bromodomain-containing protein 4 (BRD4) are currently being evaluated in clinical trials. However, the mechanisms by which BRD4 and DOT1L regulate leukemogenic transcription programs remain unclear. Using quantitative proteomics, chemoproteomics and biochemical fractionation, we found that native BRD4 and DOT1L exist in separate protein complexes. Genetic disruption or small-molecule inhibition of BRD4 and DOT1L showed marked synergistic activity against MLL leukemia cell lines, primary human leukemia cells and mouse leukemia models. Mechanistically, we found a previously unrecognized functional collaboration between DOT1L and BRD4 that is especially important at highly transcribed genes in proximity to superenhancers. DOT1L, via dimethylated histone H3 K79, facilitates histone H4 acetylation, which in turn regulates the binding of BRD4 to chromatin. These data provide new insights into the regulation of transcription and specify a molecular framework for therapeutic intervention in this disease with poor prognosis.

Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richter's syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.

B lymphoid development is initiated by the differentiation of hematopoietic stem cells into lineage committed progenitors, ultimately generating mature B cells. This highly regulated process generates clonal immunological diversity via recombination of immunoglobulin V, D and J gene segments. While several transcription factors that control B cell development and V(D)J recombination have been defined, how these processes are initiated and coordinated into a precise regulatory network remains poorly understood. Here, we show that the transcription factor ETS Related Gene (Erg) is essential for early B lymphoid differentiation. Erg initiates a transcriptional network involving the B cell lineage defining genes, Ebf1 and Pax5, which directly promotes expression of key genes involved in V(D)J recombination and formation of the B cell receptor. Complementation of Erg deficiency with a productively rearranged immunoglobulin gene rescued B lineage development, demonstrating that Erg is an essential and stage-specific regulator of the gene regulatory network controlling B lymphopoiesis.

The p53 tumor suppressor protein plays a central role in maintaining genomic integrity by occupying a nodal point in the DNA damage control pathway. Here it integrates a wide variety of signals, responding in one of several ways, that is, cell cycle arrest, senescence or programmed cell death (apoptosis). Mutations in the tumor suppressor gene tp53, which affects the key transcriptional regulatory processes in cell growth and death, occur frequently in cancer and helps explain why p53 has been called the guardian of the genome. There is a vast body of published knowledge on all aspects of p53's role in cancer. To facilitate research, it would be helpful if this information could be collected, curated and updated in a format that is easily accessible to the user community. To this end, we initiated the p53 knowledgebase project (http://p53.bii. a-star.edu.sg). The p53 knowledgebase is a user-friendly web portal incorporating visualization and analysis tools that integrates information from the published literature with other manually curated information to facilitate knowledge discovery. This includes curated information on sequence, structural, mutation, polymorphisms, protein-protein interactions, transcription factors, transcriptional targets, antibodies and post-translational modifications that involve p53. The goal is to collect and maintain all relevant data on p53 and present it in an easily accessible format that will be useful to researchers in the field.