A Trib2-p38 axis controls myeloid leukaemia cell cycle and stress response signalling

Trib2 pseudokinase is involved in the etiology of a number of cancers including leukaemia, melanoma, ovarian, lung and liver cancer. Both high and low Trib2 expression levels correlate with different types of cancer. Elevated Trib2 expression has oncogenic properties in both leukaemia and lung cancer dependent on interactions with proteasome machinery proteins and degradation of transcription factors. Here, we demonstrated that Trib2 deficiency conferred a growth and survival advantage both at steady state and in stress conditions in leukaemia cells. In response to stress, wild type leukaemia cells exited the cell cycle and underwent apoptosis. In contrast, Trib2 deficient leukaemia cells continued to enter mitosis and survive. We showed that Trib2 deficient leukaemia cells had defective MAPK p38 signalling, which associated with a reduced γ-H2Ax and Chk1 stress signalling response, and continued proliferation following stress, associated with inefficient activation of cell cycle inhibitors p21, p16 and p19. Furthermore, Trib2 deficient leukaemia cells were more resistant to chemotherapy than wild type leukaemia cells, having less apoptosis and continued propagation. Trib2 re-expression or pharmacological activation of p38 in Trib2 deficient leukaemia cells sensitised the cells to chemotherapy-induced apoptosis comparable with wild type leukaemia cells. Our data provide evidence for a tumour suppressor role of Trib2 in myeloid leukaemia via activation of p38 stress signalling. This newly identified role indicates that Trib2 may counteract the propagation and chemotherapy resistance of leukaemia cells

Age-specific biological and molecular profiling distinguishes paediatric from adult acute myeloid leukaemias

Acute myeloid leukaemia (AML) affects children and adults of all ages. AML remains one of the major causes of death in children with cancer and for children with AML relapse is the most common cause of death. Here, by modelling AML in vivo we demonstrate that AML is discriminated by the age of the cell of origin. Young cells give rise to myeloid, lymphoid or mixed phenotype acute leukaemia, whereas adult cells give rise exclusively to AML, with a shorter latency. Unlike adult, young AML cells do not remodel the bone marrow stroma. Transcriptional analysis distinguishes young AML by the upregulation of immune pathways. Analysis of human paediatric AML samples recapitulates a paediatric immune cell interaction gene signature, highlighting two genes, RGS10 and FAM26F as prognostically significant. This work advances our understanding of paediatric AML biology, and provides murine models that offer the potential for developing paediatric specific therapeutic strategies

Targeting the arginine metabolic brake enhances immunotherapy for leukaemia

Therapeutic approaches which aim to target Acute Myeloid Leukaemia through enhancement of patients’ immune responses have demonstrated limited efficacy to date, despite encouraging preclinical data. Examination of AML patients treated with azacitidine (AZA) and vorinostat (VOR) in a Phase II trial, demonstrated an increase in the expression of Cancer‐Testis Antigens (MAGE, RAGE, LAGE, SSX2 and TRAG3) on blasts and that these can be recognised by circulating antigen‐specific T cells. Although the T cells have the potential to be activated by these unmasked antigens, the low arginine microenvironment created by AML blast Arginase II activity acts a metabolic brake leading to T cell exhaustion. T cells exhibit impaired proliferation, reduced IFN‐γ release and PD‐1 up‐regulation in response to antigen stimulation under low arginine conditions. Inhibition of arginine metabolism enhanced the proliferation and cytotoxicity of anti‐NY‐ESO T cells against AZA/VOR treated AML blasts, and can boost anti‐CD33 Chimeric Antigen Receptor‐T cell cytotoxicity. Therefore, measurement of plasma arginine concentrations in combination with therapeutic targeting of arginase activity in AML blasts could be a key adjunct to immunotherapy

Investigation of the DNA damage in haematopoiesis using CRISPR/Cas9 technology

DNA replication is a highly regulated process that ensures the faithful duplication of the genome. However, endogenous or exogenous factors, such as limited DNA replication components and chemotherapeutic drugs, respectively, can cause defects in its regulation resulting in the stalling or slowing of the replication fork progression; a conditioned called replication stress (RS). Persistent RS can be detrimental to cellular survival and functions causing accumulation of DNA damage often leading to genomic instability (GI) and tumourigenesis. Haematopoiesis, a highly regulated process for blood cell generation is strongly affected by physiological ageing; molecular changes, epigenetic alterations, functional decline and abnormal cellular behaviour. These changes are often accompanied with increased DNA damage predisposing cells to leukaemic transformation. However, there is still limited data on how DNA damage and RS can affect normal haematopoiesis. This work was aimed to elucidate the effects of DNA damage in normal murine haematopoietic cells using the CRISPR/Cas9 technology to generate efficient gene knockout (KO) models. Following a simple and rapid pipeline, our CRISPR/Cas9-mediated KO models targeting a DNA replication factor, MCM6, and an anti-ageing factor also involved in DNA damage repair (DDR), SIRT1, were successfully validated for both on-target efficiency and off-target activity at the bulk population and/or single cell level. Our data showed that KO haematopoietic cells exhibited increased levels of DNA damage. This further affected haematopoietic cellular properties and functions in a way that KO cells could share some characteristics of the physiological ageing phenotype predisposing them in profound GI. Finally, another aspect of this work was to model a common chromosomal translocation found in AML patients, MLL-AF9 (MA9), using CRISPR/Cas9 technology. Several techniques and methods have been established over the years for modelling human AML either using murine or human primary cells. Each one of them have their own caveats resulting in poor recapitulation of the actual disease phenotype reported in humans. We therefore used our validated CRISPR/Cas9-system to induce double strand breaks on both chromosomes around the breakpoint cluster region to de novo generate MA9 translocation in murine haematopoietic cells. We successfully confirmed the chromosomal translocation at both DNA and mRNA levels and showed that generated MA9 translocation could confer phenotypic and molecular characteristics of the MA9+ AML cells. Altogether, this data supports that the endogenous expression of a chromosomal or genetic aberration could generate a better, more convenient model to study human diseases. Overall, this study contributes to a better understanding of the role of DNA damage in normal haematopoietic functions and reveals a potential link with ageing-associated characteristics. Finally, this study further supports that CRIPSR/Cas9 technology is a powerful tool with high efficiency, specificity and minimal off-target activity to be used for gene silencing and editing purposes

Absence of Mcm6 mimics haemopoietic ageing and has implications for leukaemic transformation

Maintenance of genomic stability is critical for tumour suppression. Deregulation of the DNA replication machinery leads to accumulation of replication stress and increased mutational events often leading to oncogenesis. Replication stress occurs during normal cell ageing; aged cells acquire more background mutations and show inefficient DNA damage repair response (DDRR). Here, we aimed to investigate whether background mutations affect haemopoietic stem and progenitor cell (HSPC) functionality and leukaemic transformation. We generated an ageing-like haemopoietic model using CRISPR lentiviral vectors to knockout (KO) Mcm6, a key factor of the DNA replication machinery whose expression decreases with age. Murine HSPCs were transduced with CRISPR empty vector (EV) or carrying guide RNAs (gRNAs) targeting Mcm6. HSPC functionality was assessed in colony forming cell assays. KO cells formed fewer, but morphologically bigger and more compact colonies indicating a defect in self-renewal/stemness. Flow cytometry analysis revealed that KO cells expressed significantly lower levels of ter119 and c-Kit markers and higher levels of CD71 suggesting a block in erythropoiesis in vitro. To elucidate Mcm6 KO effects in vivo, transduced HSPCs were injected into lethally irradiated mice. Mice that received KO cells (KO chimeras) had lower levels of CD19 B cell marker and higher CD11b and Gr-1 myeloid markers in the peripheral blood. Bone marrow cells isolated from the KO chimeric mice accumulated replication stress evident by an increase in γH2AX, and showed poor DDRR supported by the lower expression of key DDR genes. Finally, using EV and KO transduced 32D cells, we assessed leukaemic transformation following MLL-AF9 (MA9) oncogene expression. 32D-KO-MA9+ cells exhibited a proliferative advantage and expressed lower levels of the c-kit progenitor marker compared to controls indicating that DNA replication stress as a result of Mcm6 deficiency changes the leukaemic phenotype. Collectively, our data indicates that replication stress alters both haemopoietic differentiation and leukaemic transformation, reminiscent of HSPC aged cells and the increased incidence of AML in the aged

CRISPR gene editing of murine blood stem and progenitor cells induces MLL-AF9 chromosomal translocation and MLL-AF9 leukaemogenesis

Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro