To pursue a systematic approach to the discovery of functional connections among diseases, genetic perturbation, and drug action, we have created the first installment of a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules, together with pattern-matching software to mine these data. We demonstrate that this "Connectivity Map" resource can be used to find connections among small molecules sharing a mechanism of action, chemicals and physiological processes, and diseases and drugs. These results indicate the feasibility of the approach and suggest the value of a large-scale community Connectivity Map project.

To understand the role of FoxO family members in hematopoiesis, we conditionally deleted FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system. FoxO-deficient mice exhibited myeloid lineage expansion, lymphoid developmental abnormalities, and a marked decrease of the lineage-negative Sca-1+, c-Kit+ (LSK) compartment that contains the short- and long-term hematopoietic stem cell (HSC) populations. FoxO-deficient bone marrow had defective long-term repopulating activity that correlated with increased cell cycling and apoptosis of HSC. Notably, there was a marked context-dependent increase in reactive oxygen species (ROS) in FoxO-deficient HSC compared with wild-type HSC that correlated with changes in expression of genes that regulate ROS. Furthermore, in vivo treatment with the antioxidative agent N-acetyl-L-cysteine resulted in reversion of the FoxO-deficient HSC phenotype. Thus, FoxO proteins play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment, a function that is required for its long-term regenerative potential.

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL-AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.

We show that the antiapoptotic proteins BCL-2, BCL-XL, MCL-1, BFL-1, and BCL-w each bear a unique pattern of interaction with a panel of peptides derived from BH3 domains of BH3-only proteins. Cellular dependence on an antiapoptotic protein for survival can be decoded based on the pattern of mitochondrial sensitivity to this peptide panel, a strategy that we call BH3 profiling. Dependence on antiapoptotic proteins correlates with sequestration of activator BH3-only proteins like BID or BIM by antiapoptotic proteins. Sensitivity to the cell-permeable BCL-2 antagonist ABT-737 is also related to priming of BCL-2 by activator BH3-only molecules. Our data allow us to distinguish a cellular state we call "primed for death," which can be determined by BH3 profiling and which correlates with dependence on antiapoptotic family members for survival.

Acute lymphoblastic leukemias carrying a chromosomal translocation involving the mixed-lineage leukemia gene (MLL, ALL1, HRX) have a particularly poor prognosis. Here we show that they have a characteristic, highly distinct gene expression profile that is consistent with an early hematopoietic progenitor expressing select multilineage markers and individual HOX genes. Clustering algorithms reveal that lymphoblastic leukemias with MLL translocations can clearly be separated from conventional acute lymphoblastic and acute myelogenous leukemias. We propose that they constitute a distinct disease, denoted here as MLL, and show that the differences in gene expression are robust enough to classify leukemias correctly as MLL, acute lymphoblastic leukemia or acute myelogenous leukemia. Establishing that MLL is a unique entity is critical, as it mandates the examination of selectively expressed genes for urgently needed molecular targets.

Translocations that involve the mixed lineage leukaemia (MLL) gene identify a unique group of acute leukaemias, and often predict a poor prognosis. The MLL gene encodes a DNA-binding protein that methylates histone H3 lysine 4 (H3K4), and positively regulates gene expression including multiple Hox genes. Leukaemogenic MLL translocations encode MLL fusion proteins that have lost H3K4 methyltransferase activity. A key feature of MLL fusion proteins is their ability to efficiently transform haematopoietic cells into leukaemia stem cells. The link between a chromatin modulator and leukaemia stem cells provides support for epigenetic landscapes as an important part of leukaemia and normal stem-cell development.

SUMMARY Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.

Summary The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3 and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSC). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSC. Inactivation of Dot1l lead to down-regulation of direct MLL-AF9 targets and an MLL-translocation associated gene expression signature, while global gene expression remained largely unaffected. Suppression of MLL-translocation associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.