Peter P. Ruvolo

Phosphorylation of Bcl2 and regulation of apoptosis

Members of the Bcl2 family of proteins are important regulators of programmed cell death pathways with individual members that can suppress (eg Bcl2, Bcl-XL) or promote (eg Bax, Bad) apoptosis. While the mechanism(s) of Bcl2’s anti-apoptotic function is not yet clear, introduction of Bcl2 into most eukaryotic cell types will protect the recipient cell from a wide variety of stress applications that lead to cell death. There are, however, physiologic situations in which Bcl2 expression apparently fails to protect cells from apoptosis (eg negative selection of thymocytes). Further, Bcl2 expression in patient tumor samples does not consistently correlate with a worse outcome or resistance to anticancer therapies. For example, patient response and survival following chemotherapy is independent of Bcl2 expression at least for pediatric patients with ALL. These findings indicate that simple expression of Bcl2 may not be enough to functionally protect cells from apoptosis. The finding that Bcl2 is post-translationally modified by phosphorylation suggests another level of regulation of function. Recent studies have shown that agonist-activated phosphorylation of Bcl2 at serine 70 (single site phosphorylation), a site within the flexible loop domain (FLD), is required for Bcl2’s full and potent anti-apoptotic function, at least in murine IL-3-dependent myeloid cell lines. Several protein kinases have now been demonstrated to be physiologic Bcl2 kinases indicating the importance of this post-translational modification. Since Bcl2 phosphorylation has been found to be a dynamic process involving both a Bcl2 kinase(s) and phosphatase(s), a mechanism exists to rapidly and reversibly regulate Bcl2’s activity and affect cell viability. In addition, multisite Bcl2 phosphorylation induced by anti-mitotic drugs like paclitaxel may inhibit Bcl2 indicating the potential wide range of functional consequences that this post-translational modification may have on function. While post-translational mechanisms other than phosphorylation may also regulate Bcl2’s function (eg ubiquitination), this review will focus on the regulatory role for phosphorylation and discuss its potential clinical ramifications.

Novel Role for JNK as a Stress-activated Bcl2 Kinase

Interleukin (IL)-3-induced Bcl2 phosphorylation at Ser70 may be required for its full and potent antiapoptotic activity. However, in the absence of IL-3, increased expression of Bcl2 can also prolong cell survival. To determine how Bcl2 may be functionally phosphorylated following IL-3 withdrawal, astress-activated Bcl2 kinase (SAK) was sought. Results indicate that anisomycin, a potent activator of the stress kinase JNK/SAPK, can induce Bcl2 phosphorylation at Ser70 and that JNK1 can be latently activated following IL-3 withdrawal to mediate Bcl2 phosphorylation. JNK1 directly phosphorylates Bcl2 in vitro, co-localizes with Bcl2, and collaborates with Bcl-2 to mediate prolonged cell survival in the absence of IL-3 or following various stress applications. Dominant-negative (DN)-JNK1 can block both anisomycin and latent IL-3 withdrawal-induced Bcl2 phosphorylation (>90%) and potently enhances cell death. Furthermore, low dose okadaic acid (OA), a potent protein phosphatase 1 and 2A inhibitor, can activate the mitogen-activated protein kinases JNK1 and ERK1/2, but not p38 kinase, to induce Bcl2 phosphorylation and prolong cell survival in factor-deprived cells. Since PD98059, a specific MEK inhibitor, can only partially inhibit OA-induced Bcl2 phosphorylation but completely blocks OA-induced Bcl2 phosphorylation in cells expressing DN-JNK1, this supports the conclusion that OA may stimulate Bcl2 phosphorylation via a mechanism involving both JNK1 and ERK1/2. Collectively, these findings indicate a novel role for JNK1 as a SAK and may explain, at least in part, how functional phosphorylation of Bc12 can occur in the absence of growth factor.

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.

Bryostatin-I: An antineoplastic treasure from the deep?

No Abstract Available Commentary to: Title: Flavopiridol Inversely Affects p21WAF1/CIP1 and p53 and Protects p21-Sensitive Cells from Paclitaxel Authors: Mikhail V. Blagosklonny, Zbigniew Darzynkiewicz and William D. Figg Page Numbers: 420-425

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.

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.

Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-κB mediates chemoresistance

Leukemia cells are protected from chemotherapy-induced apoptosis by their interactions with bone marrow mesenchymal stromal cells (BM-MSCs). Yet the underlying mechanisms associated with this protective effect remain unclear. Genome-wide gene expression profiling of BM-MSCs revealed that coculture with leukemia cells upregulated the transcription of genes associated with nuclear factor (NF)-κB signaling. Moreover, primary BM-MSCs from leukemia patients expressed NF-κB target genes at higher levels than their normal BM-MSC counterparts. The blockade of NF-κB activation via chemical agents or the overexpression of the mutant form of inhibitor κB-α (IκBα) in BM-MSCs markedly reduced the stromal-mediated drug resistance in leukemia cells in vitro and in vivo. In particular, our unique in vivo model of human leukemia BM microenvironment illustrated a direct link between NF-κB activation and stromal-associated chemoprotection. Mechanistic in vitro studies revealed that the interaction between vascular cell adhesion molecule 1 (VCAM-1) and very late antigen-4 (VLA-4) played an integral role in the activation of NF-κB in the stromal and tumor cell compartments. Together, these results suggest that reciprocal NF-κB activation in BM-MSCs and leukemia cells is essential for promoting chemoresistance in the transformed cells, and targeting NF-κB or VLA-4/VCAM-1 signaling could be a clinically relevant mechanism to overcome stroma-mediated chemoresistance in BM-resident leukemia cells.

MEK Inhibition Enhances ABT-737-Induced Leukemia Cell Apoptosis via Prevention of ERK activated MCL-1 induction and Modulation of MCL-1/BIM Complex

Recently, strategies for acute myeloid leukemia (AML) therapy have been developed that target anti-apoptotic BCL2 family members using BH3-mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-XL, ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of the extracellular receptor activated kinase and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737-induced MCL-1 expression, and when combined with ABT-737 resulted in potent synergistic killing of AML-derived cell lines, primary AML blast and CD34+38-123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells.Whereas control mice and CI-1040-treated mice exhibited progressive leukemia growth, ABT-737, and to a significantly greater extent, ABT-737+CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Ceramide Regulates Protein Synthesis by a Novel Mechanism Involving the Cellular PKR Activator RAX

The sphingolipid ceramide is an important second signal molecule and potent apoptotic agent. The production of ceramide is associated with virtually every known stress stimulus, and thus, generation of this sphingolipid has been suggested as a universal feature of apoptosis. Recent studies suggest that an important component of cell death following diverse stress stimuli (e.g. interleukin-3 withdrawal, sodium arsenite treatment, and peroxide treatment) is the activation of the double-stranded RNA-activable protein kinase, PKR, resulting in the inhibition of protein synthesis (Ito, T., Jagus, R., and May, W. S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7455–7459). The recently discovered cellular PKR activator, RAX, is phosphorylated in association with PKR activation (Ito, T., Yang, M., and May, W. S. (1999) J. Biol. Chem. 274, 15427–15432). Since RAX is phosphorylated by an as yet undetermined SAPK and ceramide is a potent activator of SAPKs such as JNK, a role for ceramide in the activation of RAX might be possible. Results indicate that overexpression of exogenous RAX potentiates ceramide-induced killing. Furthermore, ceramide can potently inhibit protein synthesis. Since ceramide potently promotes RAX and eukaryotic initiation factor-2α phosphorylation, a possible role for ceramide in this process may involve the activation of PKR by RAX. Since 2-aminopurine, a serine/threonine kinase inhibitor that has previously been shown to inhibit PKR, blocks both the potentiation of ceramide killing by RAX and ceramide-induced inhibition of protein synthesis, ceramide appears to promote PKR activation, at least indirectly. Collectively, these findings suggest a novel role for ceramide in the regulation of protein synthesis and apoptosis.