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

Project description:Targeting the epigenome to modulate gene expression programs driving cancer development has emerged as an exciting avenue for therapeutic intervention. Pharmacological inhibition of the bromodomain and extraterminal (BET) family of chromatin adapter proteins has proven effective in this regard, suppressing growth of diverse cancer types mainly through downregulation of the c-MYC oncogene and its downstream transcriptional program. While initially effective, resistance to BET inhibitors (BETi) typically occurs through mechanisms that reactivate MYC expression. We have previously shown that lung adenocarcinoma (LAC) is inhibited by JQ1 through suppression of FOSL1, suggesting that the epigenetic landscape of tumor cells from different origins and differentiation states influences BETi response. Here, we assessed how these differences affect mechanisms of BETi resistance through the establishment of isogenic pairs of JQ1 sensitive and resistant LAC cell lines. We found that resistance to JQ1 in LAC occurs independent of FOSL1 while MYC levels remain unchanged between resistant cells and their JQ1 treated parental counterparts. Furthermore, while epithelial-mesenchymal transition (EMT) is observed upon resistance, TGF- induced EMT did not confer resistance in JQ1 sensitive LAC lines, suggesting this is a consequence, rather than a driver of BETi resistance in our model systems. Importantly, siRNA knockdown demonstrated that JQ1 resistant cell lines are still dependent on BRD4 expression and we found that phosphorylation of BRD4 is elevated in resistant LACs, identifying casein kinase 2 (CK2) as a candidate protein mediating this effect. Inhibition of CK2, as well as downstream transcriptional targets of phosphorylated BRD4 - including AXL and activators of the PI3K pathway - synergize with JQ1 to inhibit BETi resistant LAC. Overall, this demonstrates that the mechanism of resistance to BETi varies depending on cancer type, with LAC cells developing JQ1 resistance independent of MYC re-activation, and identifying CK2 phosphorylation of BRD4 as a potential target to overcome resistance in this cancer.

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Transcriptional plasticity promotes primary and acquired resistance to BET bromodomain inhibition

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