Vivian Ruvolo

Abrogation of nuclear receptors Nr4a3 andNr4a1 leads to development of acute myeloid leukemia

Nur77 (NR4A1) and Nor-1 (NR4A3) are highly homologous orphan nuclear receptors that regulate the transcription of overlapping target genes. The transcriptional activity of both proteins is regulated in a ligand-independent manner by cell- and stimulus-specific gene induction and protein phosphorylation. Nor-1 and Nur77 have been implicated in a variety of cellular processes, including the transduction of hormonal, inflammatory, mitogenic, apoptotic and differentiative signals. Cellular responses to these proteins suggest that they may function as homeostatic regulators of proliferation, apoptosis and differentiation, and thus may regulate cellular susceptibility to tumorigenesis. Their physiological functions, however, remain poorly understood. Here we describe a previously unsuspected function of Nor-1 and Nur77—as critical tumor suppressors of myeloid leukemogenesis. The abrogation of these proteins in mice led to rapidly lethal acute myeloid leukemia (AML), involving abnormal expansion of hematopoietic stem cells (HSCs) and myeloid progenitors, decreased expression of the AP-1 transcription factors JunB and c-Jun and defective extrinsic apoptotic (Fas-L and TRAIL) signaling. We found that downregulation of NR4A3 ( NOR-1 ) and NR4A1 ( NUR77 ) was a common feature in leukemic blasts from human AML patients, irrespective of karyotype. Thus Nor-1 and Nur77 may provide potential targets for therapeutic intervention in AML.

Selective BCL-2 Inhibition by ABT-199 Causes On-Target Cell Death in Acute Myeloid Leukemia

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here, we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nmol/L, and cell death occurred within 2 hours. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on-target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.

Roles of the Ras/Raf/MEK/ERK pathway in leukemia therapy

The Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is often implicated in sensitivity and resistance to leukemia therapy. Dysregulated signaling through the Ras/Raf/MEK/ERK pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Unrestricted leukemia proliferation and decreased sensitivity to apoptotic-inducing agents and chemoresistance are typically associated with activation of pro-survival pathways. Mutations in this pathway and upstream signaling molecules can alter sensitivity to small molecule inhibitors targeting components of this cascade as well as to inhibitors targeting other key pathways (for example, phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR)) activated in leukemia. Similarly, PI3K mutations can result in resistance to inhibitors targeting the Ras/Raf/MEK/ERK pathway, indicating important interaction points between the pathways (cross-talk). Furthermore, the Ras/Raf/MEK/ERK pathway can be activated by chemotherapeutic drugs commonly used in leukemia therapy. This review discusses the mechanisms by which abnormal expression of the Ras/Raf/MEK/ERK pathway can contribute to drug resistance as well as resistance to targeted leukemia therapy. Controlling the expression of this pathway could improve leukemia therapy and ameliorate human health.

Sorafenib induces apoptosis of AML cells via Bim-mediated activation of the intrinsic apoptotic pathway

Raf/MEK/Erk signaling is activated in the majority of acute myeloid leukemias (AMLs), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and proapoptotic effects of sorafenib in AML. Our studies demonstrated that sorafenib significantly inhibited the phosphorylation levels of Raf downstream target proteins MEK1/2 and Erk, induced apoptosis and inhibited colony formation in AML cell lines and in primary AML samples. Mechanistically, treatment with sorafenib resulted in upregulation of proapoptotic Bim, accompanied by an increase in Bad, Bax and Bak protein levels and decreased Mcl-1, X-linked inhibitor of apoptosis and surviving levels, which mainly led to the activation of the intrinsic apoptotic pathway. Silencing of Bim protein expression significantly abrogated sorafenib-induced apoptosis, suggesting a critical function of Bim in the activation of the intrinsic mitochondrial pathway induced by sorafenib. Importantly, sorafenib also modulated phospho-Erk, Bim, Bax and Mcl-1 levels in samples procured from patients in an ongoing Phase I clinical trial of sorafenib in AML. Combination of sorafenib with cytarabine or the novel small molecule Bcl-2 inhibitor ABT-737 synergistically induced cell death in AML cell lines. Our results strongly suggest potential activity of sorafenib as a novel mechanism-based therapeutic agent in AML.

Targeting the translational apparatus to improve leukemia therapy: roles of the PI3K/PTEN/Akt/mTOR pathway

It has become apparent that regulation of protein translation is an important determinant in controlling cell growth and leukemic transformation. The phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway is often implicated in sensitivity and resistance to therapy. Dysregulated signaling through the PI3K/PTEN/Akt/mTOR pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Furthermore, this pathway is activated by autocrine transformation mechanisms. PTEN is a critical tumor suppressor gene and its dysregulation results in the activation of Akt. PTEN is often mutated, silenced and is often haploinsufficient. The mTOR complex1 (mTORC1) regulates the assembly of the eukaryotic initiation factor4F complex, which is critical for the translation of mRNAs that are important for cell growth, prevention of apoptosis and transformation. These mRNAs have long 5′-untranslated regions that are G+C rich, rendering them difficult to translate. Elevated mTORC1 activity promotes the translation of these mRNAs via the phosphorylation of 4E-BP1. mTORC1 is a target of rapamycin and novel active-site inhibitors that directly target the TOR kinase activity. Although rapamycin and novel rapalogs are usually cytostatic and not cytotoxic for leukemic cells, novel inhibitors that target the kinase activities of PI3K and mTOR may prove more effective for leukemia therapy.

Roles of the RassRafsMEKsERK pathway in leukemia therapy

The Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is often implicated in sensitivity and resistance to leukemia therapy. Dysregulated signaling through the Ras/Raf/MEK/ERK pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Unrestricted leukemia proliferation and decreased sensitivity to apoptotic-inducing agents and chemoresistance are typically associated with activation of pro-survival pathways. Mutations in this pathway and upstream signaling molecules can alter sensitivity to small molecule inhibitors targeting components of this cascade as well as to inhibitors targeting other key pathways (for example, phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR)) activated in leukemia. Similarly, PI3K mutations can result in resistance to inhibitors targeting the Ras/Raf/MEK/ERK pathway, indicating important interaction points between the pathways (cross-talk). Furthermore, the Ras/Raf/MEK/ERK pathway can be activated by chemotherapeutic drugs commonly used in leukemia therapy. This review discusses the mechanisms by which abnormal expression of the Ras/Raf/MEK/ERK pathway can contribute to drug resistance as well as resistance to targeted leukemia therapy. Controlling the expression of this pathway could improve leukemia therapy and ameliorate human health.

The novel triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid inhibits metastatic murine breast tumor growth through inactivation of STAT3 signaling

We and others have reported that C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) effectively inhibits the growth of multiple cancer cell types. Our previous studies indicated that prolonged CDDO-Me treatment inactivated extracellular signal-regulated kinase signaling in acute myelogenous leukemia cells. Whether treatment with CDDO-Me has an earlier effect on other proteins that are important for either signal transduction or oncogenesis is unknown. Constitutively activated signal transducer and activator of transcription 3 (STAT3) is frequently found in human breast cancer samples. Constitutively activated STAT3 was shown to up-regulate c-Myc in several types of cancer and has a feedback effect on Src and Akt. To examine the effects of CDDO-Me on STAT3 signaling in breast cancer, we used the murine 4T1 breast tumor model, which is largely resistant to chemotherapy. In vitro, after treatment of 4T1 cells with 500 nmol/L CDDO-Me for 2 h, we found (a) inactivation of STAT3, (b) inactivation of Src and Akt, (c) 4-fold reduction of c-Myc mRNA levels, (d) accumulation of cells in G(2)-M cell cycle phase, (e) abrogation of invasive growth of 4T1 cells, and (f) lack of apoptosis induction. In in vivo studies, CDDO-Me completely eliminated 4T1 breast cancer growth and lung metastases induced by 4T1 cells in mice when treatment started 1 day after tumor implantation and significantly inhibited tumor growth when started after 5 days. In vivo studies also indicated that splenic mature dendritic cells were restored after CDDO-Me treatment. In summary, these data suggest that CDDO-Me may have therapeutic potential in breast cancer therapy, in part, through inactivation of STAT3.

The dual PI3 kinase/mTOR inhibitor PI-103 prevents p53 induction by Mdm2 inhibition but enhances p53-mediated mitochondrial apoptosis in p53 wild-type AML

Activation of the phosphatidylinositol-3 kinase/Akt/mammalian target of the rapamycin (PI3K/Akt/mTOR) pathway and inactivation of wild-type p53 by murine double minute 2 homologue (Mdm2) overexpression are frequent molecular events in acute myeloid leukemia (AML). We investigated the interaction of PI3K/Akt/mTOR and p53 pathways after their simultaneous blockade using the dual PI3K/mTOR inhibitor PI-103 and the Mdm2 inhibitor Nutlin-3. We found that PI-103, which itself has modest apoptogenic activity, acts synergistically with Nutlin-3 to induce apoptosis in a wild-type p53-dependent fashion. PI-103 synergized with Nutlin-3 to induce Bax conformational change and caspase-3 activation, despite its inhibitory effect on p53 induction. The PI-103/Nutlin-3 combination caused profound dephosphorylation of 4E-BP1 and decreased expression of many proteins including Mdm2, p21, Noxa, Bcl-2 and survivin, which can affect mitochondrial stability. We suggest that PI-103 actively enhances downstream p53 signaling and that a combination strategy aimed at inhibiting PI3K/Akt/mTOR signaling and activating p53 signaling is potentially effective in AML, where TP53 mutations are rare and downstream p53 signaling is intact.

Prognostic impact and targeting of CRM1 in acute myeloid leukemia

Chromosomal region maintenance 1 (CRM1) is a nuclear export receptor recognizing proteins bearing a leucine-rich nuclear export signal. CRM1 is involved in nuclear export of tumor suppressors such as p53. We investigated the prognostic significance of CRM1 in acute myeloid leukemia (AML) and effects of a novel small-molecule selective inhibitor of CRM1. CRM1 protein expression was determined in 511 newly diagnosed AML patients and was correlated with mouse double minute 2 (MDM2) and p53 levels. High CRM1 expression was associated with short survival of patients and remained an adverse prognostic factor in multivariate analysis. CRM1 inhibitor KPT-185 induced mainly full-length p53 and apoptosis in a p53-dependent manner, whereas inhibition of proliferation was p53 independent. Patient samples with p53 mutations showed low sensitivity to KPT-185. Nuclear retention of p53 induced by CRM1 inhibition synergized with increased levels of p53 induced by MDM2 inhibition in apoptosis induction. KPT-185 and Nutlin-3a, alone and in combination, induced synergistic apoptosis in patient-derived CD34(+)/CD38(-) AML, but not in normal progenitor cells. Data suggest that CRM1 exerts an antiapoptotic function and is highly prognostic in AML. We propose a novel combinatorial approach for the therapy of AML, aimed at maximal activation of p53-mediated apoptosis by concomitant MDM2 and CRM1 inhibition.

HOX expression patterns identify a common signature for favorable AML.

Deregulated HOX expression, by chromosomal translocations and myeloid-lymphoid leukemia (MLL) rearrangements, is causal in some types of leukemia. Using real-time reverse transcription-PCR, we examined the expression of 43 clustered HOX, polycomb, MLL and FLT3 genes in 119 newly diagnosed adult acute myeloid leukemias (AMLs) selected from all major cytogenetic groups. Downregulated HOX expression was a consistent feature of favorable AMLs and, among these cases, inv(16) cases had a distinct expression profile. Using a 17-gene predictor in 44 additional samples, we observed a 94.7% specificity for classifying favorable vs intermediate/unfavorable cytogenetic groups. Among other AMLs, HOX overexpression was associated with nucleophosmin (NPM) mutations and we also identified a phenotypically similar subset with wt-NPM. In many unfavorable and other intermediate cytogenetic AMLs, HOX levels resembled those in normal CD34+ cells, except that the homogeneity characteristic of normal samples was not present. We also observed that HOXA9 levels were significantly inversely correlated with survival and that BMI-1 was overexpressed in cases with 11q23 rearrangements, suggesting that p19ARF suppression may be involved in MLL-associated leukemia. These results underscore the close relationship between HOX expression patterns and certain forms of AML and emphasize the need to determine whether these differences play a role in the disease process.