Treatment of chronic myeloid leukemia (CML) with imatinib mesylate and other second/third generation c-Abl specific tyrosine kinase inhibitors (TKIs) has significantly extended patient survival1. However, TKIs primarily target differentiated cells and do not eliminate leukemic stem cells (LSCs)2–4. Therefore, targeting minimal residual disease, to prevent acquired resistance and/or disease relapse requires identification of novel LSC-selective target(s) that can be exploited therapeutically5,6. Given that malignant transformation involves cellular metabolic changes, which may in turn render the transformed cells susceptible to specific assaults in a selective manner7, we searched for such vulnerabilities in CML LSCs. We performed metabolic analyses on both stem cell-enriched (CD34+ and CD34+CD38-) and differentiated (CD34-) patient derived CML cells, and compared their signature with that of normal counterparts. Combining stable isotope-assisted metabolomics with functional assays, we demonstrate that primitive CML cells rely on upregulated oxidative metabolism for their survival. We also show that combination-treatment of imatinib with tigecycline, an antibiotic that inhibits mitochondrial protein translation, selectively eradicates CML LSCs, both in vitro and in a xenotransplantation model of human CML. Our findings provide a strong indication for investigating the employment of TKIs in combination with tigecycline to treat CML patients with minimal residual disease.

SummaryChronic myeloid leukaemia (CML) arises following transformation of a haemopoietic stem cell (HSC) by protein-tyrosine kinase BCR-ABL1. Direct inhibition of BCR-ABL1 kinase has revolutionized disease management, but fails to eradicate leukaemic stem cells (LSC), which maintain CML. LSC are independent of BCR-ABL1 for survival, providing a rationale to identify and target kinase-independent pathways. Here we show using proteomics, transcriptomics and network analyses, that in human LSC aberrantly expressed proteins, in both imatinib-responder and non-responder patients are modulated in concert with p53 and c-Myc regulation. Perturbation of both p53 and c-Myc, not BCR-ABL1 itself, leads to synergistic kill, differentiation and near elimination of transplantable human LSC in mice, whilst sparing normal HSC. This unbiased systems approach targeting connected nodes exemplifies a novel precision medicine strategy providing evidence that LSC can be eradicated.

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs.

A major obstacle to curing chronic myeloid leukemia (CML) is residual disease maintained by tyrosine kinase inhibitor (TKI)-persistent leukemic stem cells (LSC). These are BCR-ABL1 kinase independent, refractory to apoptosis, and serve as a reservoir to drive relapse or TKI resistance. We demonstrate that Polycomb Repressive Complex 2 is misregulated in chronic phase CML LSCs. This is associated with extensive reprogramming of H3K27me3 targets in LSCs, thus sensitizing them to apoptosis upon treatment with an EZH2-specifi c inhibitor (EZH2i). EZH2i does not impair normal hematopoietic stem cell survival. Strikingly, treatment of primary CML cells with either EZH2i or TKI alone caused signifi cant upregulation of H3K27me3 targets, and combined treatment further potentiated these effects and resulted in signifi cant loss of LSCs compared to TKI alone, in vitro , and in long-term bone marrow murine xenografts. Our fi ndings point to a promising epigenetic-based therapeutic strategy to more effectively target LSCs in patients with CML receiving TKIs. SIGNIFICANCE:In CML, TKI-persistent LSCs remain an obstacle to cure, and approaches to eradicate them remain a signifi cant unmet clinical need. We demonstrate that EZH2 and H3K27me3 reprogramming is important for LSC survival, but renders LSCs sensitive to the combined effects of EZH2i and TKI. This represents a novel approach to more effectively target LSCs in patients receiving TKI treatment. Cancer Discov; 6(11); 1248-57.

Since publication, three errors have been highlighted:(1) Two fetal recordings were excluded from the final sample due to technical issues resulting in corruption of the scan at the source. The final sample and analysis remains unchanged. (2) A typographical error in the reported equation from Del Giudice ( 2011) was incorporated into the ''Method Details'' section.The actual calculation utilized in the original paper was the correct equation from Del Giudice (2011). (3) A statement related to normal distribution of the data has been removed from the ''Quantification and Statistical Analysis'' section. The original paper used non-parametric analyses, which are unaffected.

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: LinSca-1c-kitCD48CD150) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence.

Key Points• Hif-2a-dependent signaling is dispensable for steady-state multilineage hematopoiesis.• Hif-2a is not essential for HSC self-renewal.Local hypoxia in hematopoietic stem cell (HSC) niches is thought to regulate HSC functions. Hypoxia-inducible factor-1 (Hif-1) and Hif-2 are key mediators of cellular responses to hypoxia. Although oxygen-regulated a-subunits of Hifs, namely Hif-1a and Hif-2a, are closely related, they play overlapping and also distinct functions in nonhematopoietic tissues. Although Hif-1a-deficient HSCs lose their activity on serial transplantation, the role for Hif-2a in cell-autonomous HSC maintenance remains unknown. Here, we demonstrate that constitutive or inducible hematopoiesis-specific Hif-2a deletion does not affect HSC numbers and steady-state hematopoiesis. Furthermore, using serial transplantations and 5-fluorouracil treatment, we demonstrate that HSCs do not require Hif-2a to self-renew and recover after hematopoietic injury. Finally, we show that Hif-1a deletion has no major impact on steady-state maintenance of Hif2a-deficient HSCs and their ability to repopulate primary recipients, indicating that Hif-1a expression does not account for normal behavior of Hif-2a-deficient HSCs. (Blood. 2013;122(10):1741-1745

People with Down syndrome are living longer but still die younger of different causes than the general population. More robust comparative data are needed, and ethnic differences require further study.