Matthew R. Janes

The translational landscape of mTOR signalling steers cancer initiation and metastasis

The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth and cancer. However, the downstream translationally regulated nodes of gene expression that may direct cancer development are poorly characterized. Using ribosome profiling, we uncover specialized translation of the prostate cancer genome by oncogenic mTOR signalling, revealing a remarkably specific repertoire of genes involved in cell proliferation, metabolism and invasion. We extend these findings by functionally characterizing a class of translationally controlled pro-invasion messenger RNAs that we show direct prostate cancer invasion and metastasis downstream of oncogenic mTOR signalling. Furthermore, we develop a clinically relevant ATP site inhibitor of mTOR, INK128, which reprograms this gene expression signature with therapeutic benefit for prostate cancer metastasis, for which there is presently no cure. Together, these findings extend our understanding of how the ‘cancerous’ translation machinery steers specific cancer cell behaviours, including metastasis, and may be therapeutically targeted.

Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor

Targeting the mammalian target of rapamycin (mTOR) protein is a promising strategy for cancer therapy. The mTOR kinase functions in two complexes, TORC1 (target of rapamycin complex-1) and TORC2 (target of rapamycin complex-2); however, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. We compared rapamycin with PP242, an inhibitor of the active site of mTOR in both TORC1 and TORC2 (hereafter referred to as TORC1/2), in models of acute leukemia harboring the Philadelphia chromosome (Ph) translocation. We demonstrate that PP242, but not rapamycin, causes death of mouse and human leukemia cells. In vivo, PP242 delays leukemia onset and augments the effects of the current front-line tyrosine kinase inhibitors more effectively than does rapamycin. Unexpectedly, PP242 has much weaker effects than rapamycin on the proliferation and function of normal lymphocytes. PI-103, a less selective TORC1/2 inhibitor that also targets phosphoinositide 3-kinase (PI3K), is more immunosuppressive than PP242. These findings establish that Ph+ transformed cells are more sensitive than normal lymphocytes to selective TORC1/2 inhibitors and support the development of such inhibitors for leukemia therapy.

Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL + leukemia cells

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph+ ALL.

Efficacy of the investigational mTOR kinase inhibitor MLN0128 / INK128 in models of B-cell acute lymphoblastic leukemia

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anticancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here, we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib (DA) in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of DA. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

Targeting of the MNK–eIF4E axis in blast crisis chronic myeloid leukemia inhibits leukemia stem cell function

Significance Cancer stem cells (CSCs) frequently acquire the ability to self-renew and persist in their hosts by coopting normal stem cell programs. Blast crisis (BC) chronic myeloid leukemia is a prototypic example, as the acquired activation of β-catenin signaling that enables BC CSC function is also important in normal hematopoietic stem cell maintenance. In identifying eIF4E phosphorylation by the MNK kinases as a necessary step in β-catenin activation in BC CSCs, but not normal hematopoietic stem cells, we define a therapeutic target in BC. Our studies suggest that clinical trials with MNK kinase inhibitors are warranted in BC chronic myeloid leukemia. Chronic myeloid leukemia responds well to therapy targeting the oncogenic fusion protein BCR-ABL1 in chronic phase, but is resistant to treatment after it progresses to blast crisis (BC). BC is characterized by elevated β-catenin signaling in granulocyte macrophage progenitors (GMPs), which enables this population to function as leukemia stem cells (LSCs) and act as a reservoir for resistance. Because normal hematopoietic stem cells (HSCs) and LSCs depend on β-catenin signaling for self-renewal, strategies to specifically target BC will require identification of drugable factors capable of distinguishing between self-renewal in BC LSCs and normal HSCs. Here, we show that the MAP kinase interacting serine/threonine kinase (MNK)-eukaryotic translation initiation factor 4E (eIF4E) axis is overexpressed in BC GMPs but not normal HSCs, and that MNK kinase-dependent eIF4E phosphorylation at serine 209 activates β-catenin signaling in BC GMPs. Mechanistically, eIF4E overexpression and phosphorylation leads to increased β-catenin protein synthesis, whereas MNK-dependent eIF4E phosphorylation is required for nuclear translocation and activation of β-catenin. Accordingly, we found that a panel of small molecule MNK kinase inhibitors prevented eIF4E phosphorylation, β-catenin activation, and BC LSC function in vitro and in vivo. Our findings identify the MNK–eIF4E axis as a specific and critical regulator of BC self-renewal, and suggest that pharmacologic inhibition of the MNK kinases may be therapeutically useful in BC chronic myeloid leukemia.

Sphingolipid-based drugs selectively kill cancer cells by down-regulating nutrient transporter proteins

Cancer cells are hypersensitive to nutrient limitation because oncogenes constitutively drive glycolytic and TCA (tricarboxylic acid) cycle intermediates into biosynthetic pathways. As the anaplerotic reactions that replace these intermediates are fueled by imported nutrients, the cancer cells ability to generate ATP becomes compromised under nutrient-limiting conditions. In addition, most cancer cells have defects in autophagy, the catabolic process that provides nutrients from internal sources when external nutrients are unavailable. Normal cells, in contrast, can adapt to the nutrient stress that kills cancer cells by becoming quiescent and catabolic. In the present study we show that FTY720, a water-soluble sphingolipid drug that is effective in many animal cancer models, selectively starves cancer cells to death by down-regulating nutrient transporter proteins. Consistent with a bioenergetic mechanism of action, FTY720 induced homoeostatic autophagy. Cells were protected from FTY720 by cell-permeant nutrients or by reducing nutrient demand, but blocking apoptosis was ineffective. Importantly, AAL-149, a FTY720 analogue that lacks FTY720s dose-limiting toxicity, also triggered transporter loss and killed patient-derived leukaemias while sparing cells isolated from normal donors. As they target the metabolic profile of cancer cells rather than specific oncogenic mutations, FTY720 analogues such as AAL-149 should be effective against many different tumour types, particularly in combination with drugs that inhibit autophagy.

Immune Regulation by Rapamycin: Moving Beyond T Cells

Rapamycin regulates immune responses, in part, through its effects on macrophages and dendritic cells. The mammalian target of rapamycin (mTOR) is a multifunctional kinase that promotes cell growth and division in response to growth factor and nutrient signals. Rapamycin exerts its potent immunosuppressive effects in part through direct effects on antigen-specific lymphocytes; however, rapamycin also modulates adaptive immunity through its effects on innate immune cells, including dendritic cells and macrophages. Studies have established rapamycin-sensitive functions of mTOR, downstream of Toll-like receptors, in shaping the cytokine response of myeloid cells and driving the production of interferon by plasmacytoid dendritic cells. These findings point to new strategies for boosting or suppressing specific immune responses.

Resistance to mTOR kinase inhibitors in lymphoma cells lacking 4EBP1.

Inhibitors of the mechanistic target of rapamycin (mTOR) hold promise for treatment of hematological malignancies. Analogs of the allosteric mTOR inhibitor rapamycin are approved for mantle cell lymphoma but have limited efficacy in other blood cancers. ATP-competitive “active-site” mTOR inhibitors produce more complete mTOR inhibition and are more effective than rapamycin in preclinical models of leukemia, lymphoma and multiple myeloma. In parallel to clinical trials of active-site mTOR inhibitors, it will be important to identify resistance mechanisms that might limit drug efficacy in certain patients. From a panel of diffuse large B-cell lymphoma cell lines, we found that the VAL cell line is particularly resistant to apoptosis in the presence of active-site mTOR inhibitors. Mechanistic investigation showed that VAL does not express eukaryotic initiation factor 4E-binding protein-1 (4EBP1), a key negative regulator of translation controlled by mTOR. Although VAL cells express the related protein 4EBP2, mTOR inhibitor treatment fails to displace eukaryotic initiation factor 4G from the mRNA cap-binding complex. Knockdown of eukaryotic initiation factor 4E, or re-expression of 4EBP1, sensitizes cells to apoptosis when treated with active-site mTOR inhibitors. These findings provide a naturally occurring example of 4EBP deficiency driving lymphoma cell resistance to active-site mTOR inhibitors.

Next-generation flow cytometry

Mass cytometry dramatically enhances the dimensionality of fluorescence-based flow cytometry for phenotypic analysis of heterogeneous cell populations.

B-1 Cell Lymphoma in Mice Lacking the Steroid and Xenobiotic Receptor, SXR

The steroid and xenobiotic receptor (SXR) is a broad-specificity nuclear hormone receptor that is highly expressed in the liver and intestine, where its primary function is to regulate drug and xenobiotic metabolism. SXR is expressed at lower levels in other tissues, where little is known about its physiological functions. We previously linked SXR with immunity and inflammation by showing that SXR antagonizes the activity of nuclear factor (NF)-κB in vitro and in vivo. SXR(-/-) mice demonstrate aberrantly high NF-κB activity and overexpression of NF-κB target genes. Here we show that SXR(-/-) mice develop B cell lymphoma in an age-dependent manner. SXR(-/-) mice develop multiple hyperplastic lymphoid foci composed of B-1a cells in the intestine, spleen, lymph nodes, peritoneal cavity, and blood. In all circumstances, these lymphocytes possess cell surface and molecular characteristics of either chronic lymphocytic leukemia or non-Hodgkins lymphoma originating from B-1 lymphocytes. These results demonstrate a novel and unsuspected role for SXR signaling in the B-1 cell compartment, establish SXR as a tumor suppressor in B-1 cells, and may provide a link between metabolism of xenobiotic compounds and lymphomagenesis.