Project description:Patients with acute myeloid leukaemia (AML) often achieve remission yet subsequently die of disease relapse, which is commonly driven by chemotherapy-resistant leukaemic stem cells (LSCs). LSCs are defined by their capacity to initiate leukaemia in immuno-compromised mice. This precludes an analysis of their interaction with lymphocytes as components of anti-tumour immunity, which LSCs must escape in order to induce cancer7. Here we propose that stemness and immune evasion are closely intertwined in human AML. Using human AML xenograft and syngeneic mouse leukemia models, we show that ligands of the danger detector NKG2D, a critical mediator of anti-tumour immunity by cytotoxic lymphocytes such as natural killer (NK) cells, are generally expressed on bulk AML cells but not on LSCs. Functional LSCs can be isolated by their lack of NKG2D ligand (NKG2DL) expression, even in human AMLs not expressing the conventional LSC marker CD34. NKG2DL expressing AML cells are cleared by NK cells while NKG2DLneg leukaemic cells isolated from the same individuals escape NK cell killing. These NKG2DLneg AML cells show an immature morphology, display molecular and functional stemness characteristics, can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplants (PDX). Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) negatively regulates NKG2DL and PARP1 inhibition (PARPi) induces NKG2DL surface expression in LSCs. Consequently, co-treatment with PARPi and NK cells suppresses leukaemogenesis in PDX models. Low expression of surface NKG2DL as well as high PARP1 expression are associated with inferior clinical outcome in patients with AML. In summary, our data link the concept of LSCs to immune escape and indicate absence of immunostimulatory NKG2DL as a novel method to identify LSCs across genetic AML subtypes. Moreover, we provide a strong rationale for targeting therapy resistant LSCs by PARP1 inhibition rendering them amenable to NK cell control in vivo.

Project description:Bromodomain and Extra Terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic paradigm by directly targeting epigenetic readers1,2. Early clinical trials have shown significant promise especially in acute myeloid leukaemia (AML)3; therefore the evaluation of resistance mechanisms, an inevitable consequence of cancer therapies, is of utmost importance to optimise the clinical efficacy of these drugs. Using primary murine stem and progenitor cells immortalised with MLL-AF9, we have used an innovative approach to generate 20 cell lines derived from single cell clones demonstrating stable resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism but is demonstrated to emerge from leukaemia stem cells (LSC). Resistant clones display a leukaemic granulocyte-macrophage progenitor (L-GMP) phenotype (Lin-, Sca-, cKit+, CD34+, Fc³RII/RIII+) and functionally exhibit increased clonogenic capacity in vitro and markedly shorter leukaemia latency in vivo. Chromatin bound BRD4 is globally reduced in resistant cells, however expression of key target genes such as MYC remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors is in part a consequence of increased Wnt/²-catenin signaling. Negative regulation of this pathway results in differentiation of resistant cells into mature leukaemic blasts, inhibition of MYC expression and restoration of sensitivity to I-BET in vitro and in vivo. Finally, we show that the sensitivity of primary human AML cells to I-BET correlates with the baseline expression of Wnt/²-catenin target genes. Together these findings provide novel insights into the biology of AML, highlight the potential therapeutic limitations of BET inhibitors and identify strategies that may overcome resistance and enhance the clinical utility of these unique targeted therapies. Comparison of iBET resistant and sensitive cell lines

Project description:Bromodomain and Extra Terminal protein (BET) inhibitors are first-in-class targeted therapies that deliver a new therapeutic paradigm by directly targeting epigenetic readers. Early clinical trials have shown significant promise especially in acute myeloid leukaemia (AML)3; therefore the evaluation of resistance mechanisms, an inevitable consequence of cancer therapies, is of utmost importance to optimise the clinical efficacy of these drugs. Using primary murine stem and progenitor cells immortalised with MLL-AF9, we have used an innovative approach to generate 20 cell lines derived from single cell clones demonstrating stable resistance, in vitro and in vivo, to the prototypical BET inhibitor, I-BET. Resistance to I-BET confers cross-resistance to chemically distinct BET inhibitors such as JQ1, as well as resistance to genetic knockdown of BET proteins. Resistance is not mediated through increased drug efflux or metabolism but is demonstrated to emerge from leukaemia stem cells (LSC). Resistant clones display a leukaemic granulocyte-macrophage progenitor (L-GMP) phenotype (Lin-, Sca-, cKit+, CD34+, FcγRII/RIII+) and functionally exhibit increased clonogenic capacity in vitro and markedly shorter leukaemia latency in vivo. Chromatin bound BRD4 is globally reduced in resistant cells, however expression of key target genes such as MYC remains unaltered, highlighting the existence of alternative mechanisms to regulate transcription. We demonstrate that resistance to BET inhibitors is in part a consequence of increased Wnt/β-catenin signaling. Negative regulation of this pathway results in differentiation of resistant cells into mature leukaemic blasts, inhibition of MYC expression and restoration of sensitivity to I-BET in vitro and in vivo. Finally, we show that the sensitivity of primary human AML cells to I-BET correlates with the baseline expression of Wnt/β-catenin target genes. Together these findings provide novel insights into the biology of AML, highlight the potential therapeutic limitations of BET inhibitors and identify strategies that may overcome resistance and enhance the clinical utility of these unique targeted therapies. Total RNA obtained from iBET resistant and sensitive cells

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Mediator kinase inhibition further activates super-enhancer-associated genes in AML

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