Jason A. Powell

Targeting acute myeloid leukemia by dual inhibition of PI3K signaling and Cdk9-mediated Mcl-1 transcription

Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110α isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

The protein kinase activity of PI3K phosphorylates specific serine residues in growth factor receptors to promote cell survival; these events are constitutively activated in some leukemias.

Targeting sphingosine kinase 1 induces MCL1 dependent cell death in acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1-phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. Here we show that SPHK1 is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death in AML cell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34+ progenitors from healthy donors. Furthermore, administration of SPHK1 inhibitors to orthotopic AML patient-derived xenografts reduced tumor burden and prolonged overall survival without affecting murine hematopoiesis. SPHK1 inhibition was associated with reduced survival signaling from S1P receptor 2, resulting in selective downregulation of the prosurvival protein MCL1. Subsequent analysis showed that the combination of BH3 mimetics with either SPHK1 inhibition or S1P receptor 2 antagonism triggered synergistic AML cell death. These results support the notion that SPHK1 is a bona fide therapeutic target for the treatment of AML.

Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells

BackgroundHIV-1 reverse transcriptase (RT) is a heterodimer composed of p66 and p51 subunits and is responsible for reverse transcription of the viral RNA genome into DNA. RT can be post-translationally modified in vitro which may be an important mechanism for regulating RT activity. Here we report detection of different p66 and p51 RT isoforms by 2D gel electrophoresis in virions and infected cells.ResultsMajor isoforms of the p66 and p51 RT subunits were observed, with pIs of 8.44 and 8.31 respectively (p668.44 and p518.31). The same major isoforms were present in virions, virus-infected cell lysates and intracellular reverse transcription complexes (RTCs), and their presence in RTCs suggested that these are likely to be the forms that function in reverse transcription. Several minor RT isoforms were also observed. The observed pIs of the RT isoforms differed from the pI of theoretical unmodified RT (p668.53 and p518.60), suggesting that most of the RT protein in virions and cells is post-translationally modified. The modifications of p668.44 and p518.31 differed from each other indicating selective modification of the different RT subunits. The susceptibility of RT isoforms to phosphatase treatment suggested that some of these modifications were due to phosphorylation. Dephosphorylation, however, had no effect on in vitro RT activity associated with virions, infected cells or RTCs suggesting that the phospho-isoforms do not make a major contribution to RT activity in an in vitro assay.ConclusionThe same major isoform of p66 and p51 RT is found in virions, infected cells and RTCs and both of these subunits are post-translationally modified. This post-translational modification of RT may be important for the function of RT inside the cell.

Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.

Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase Cϵ Regulates Ras/Mitogen-activated Protein Kinase Signaling and Neuronal Differentiation

Background: The FGF receptors (FGFRs) regulate pleiotropic (diverse) cellular responses. Results: Serine 779 phosphorylation of FGFR1 and 2 by PKCϵ promotes maximal Ras/MAPK signaling and neuronal differentiation. Conclusion: The FGFRs quantitatively control signal transduction via receptor serine phosphorylation. Significance: Receptor tyrosine kinases couple to specific downstream signaling pathways via phosphoserine docking sites to control pleiotropic cellular responses. The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser779 in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser779 in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser779 was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser779 in vitro, whereas overexpression of PKCϵ results in constitutive Ser779 phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser779 phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser779, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.

Phosphorylation of Serine 779 in Fibroblast Growth Factor Receptor 1 and 2 by Protein Kinase C epsilon regulates Ras/MAP kinase signaling and neuronal differentiation

Background: The FGF receptors (FGFRs) regulate pleiotropic (diverse) cellular responses. Results: Serine 779 phosphorylation of FGFR1 and 2 by PKCϵ promotes maximal Ras/MAPK signaling and neuronal differentiation. Conclusion: The FGFRs quantitatively control signal transduction via receptor serine phosphorylation. Significance: Receptor tyrosine kinases couple to specific downstream signaling pathways via phosphoserine docking sites to control pleiotropic cellular responses. The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser779 in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser779 in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser779 was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser779 in vitro, whereas overexpression of PKCϵ results in constitutive Ser779 phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser779 phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser779, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.

The Emerging Role of Sphingolipids in Cancer Stem Cell Biology

Tumours are widely considered to be composed of a heterogeneous population of cells that collectively constitute a “community” that permits tumour cell growth. As a part of this “community”, the influx of invading immune, endothelial, and mesenchymal cells can regulate tumour growth in both positive and negative manners. Even within the tumour cell population itself, there is genetic diversity that divides the overall population into distinct sub-populations. Many chemotherapeutic regimes are effective at targeting the bulk of the tumour population. Despite the apparent eradication of the tumour population, minimal residual disease persists driving disease relapse and the subsequent treatment of relapsed/refractory disease remains a problem. A part of this problem remains the inability of current drug treatment regimens to effectively target the cancer stem cell population. Currently, there is emerging evidence to suggest that sphingolipids and alterations in sphingolipid metabolism contribute to cancer stem cell biology. Here, we summarise these findings and identify potential areas for further investigation.

Abstract A19: The selective targeting of cell survival pathways in leukemia

Drugs that selectively block cell survival pathways in malignant cells may lead to improved therapeutic outcomes. Our previous studies have identified a tyrosine kinase-independent signalling mechanism that regulates cell survival but not other cellular responses such as proliferation. We refer to this ability of some cells to survive in a largely quiescent (or dormant) state as the “survival-only” response and have shown that constitutive activation of survival-only pathways renders quiescent acute myeloid leukemia (AML) cells highly refractory to cytotoxic therapies. Thus, identifying therapeutic targets within survival-only pathways may provide new clinical approaches in the treatment of AML and possibly other cancers. We have performed both biochemical and functional screens using kinase inhibitors and siRNA to identify kinases that regulate survival-only responses in leukemic cells. These screens identified 7 “survival kinases” (Pim2, Cdk4, Cdk9, PKCe, AurK, CK2 and PI3K) that when subjected to siRNA knockdown in primary human AML cells resulted in a significant reduction in cell survival (p 70% apoptosis) but not in purified human CD34+ bone marrow progenitors from normal donors (p<0.05). Our findings provide a novel and rational approach for identifying previously unsuspected vulnerabilities in cancer cells through the dual inhibition of distinct and independent survival pathways. They also reveal previously unrecognized alliances between evolutionarily divergent kinases in promoting tyrosine kinase-independent survival-only responses and show that inhibition of multiple survival arms in AML cells is highly synergistic.nnCitation Format: Daniel Thomas, Jason A. Powell, Michael W. Parker, Paul G. Ekert, Richard B. Lock, David CS Huang, Susie K. Nilsson, Christian Recher, Andrew H. Wei, Mark A. Guthridge. The selective targeting of cell survival pathways in leukemia. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A19.