GSK-3 as a novel prognostic indicator in leukemia

Advances in Biological Regulation xxx (2017) 1e10

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GSK-3 as a novel prognostic indicator in leukemia Peter P. Ruvolo Department of Leukemia, Unit 448, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, United States

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Article history: Received 25 April 2017 Received in revised form 5 May 2017 Accepted 7 May 2017 Available online xxx

While leukemias represent a diverse set of diseases with malignant cells derived from myeloid or lymphoid origin, a common feature is the dysregulation of signal transduction pathways that influence leukemogeneisis, promote drug resistance, and favor leukemia stem cells. Mutations in PI3K, PTEN, RAS, or other upstream regulators can activate the AKT kinase which has central roles in supporting cell proliferation and survival. A major target of AKT is Glycogen Synthase Kinase 3 (GSK3). GSK3 has two isoforms (alpha and beta) that were studied as regulators of metabolism but emerged as central players in cancer in the early 1990s. GSK3 is unique in that the isoforms are constitutively active. Active GSK3 promotes destruction of oncogenic proteins such as beta Catenin, c-MYC, and MCL-1 and thus has tumor suppressor properties. In AML, inactivation of GSK3 is associated with poor overall survival. Interestingly in some leukemias GSK3 targets a tumor suppressor and thus the kinases can act as tumor promoters in those instances. An example is GSK3 targeting p27Kip1 in AML with MLL translocation. This review will cover the role of GSK3 in various leukemias both as tumor suppressor and tumor promoter. We will also briefly cover current state of GSK3 inhibitors for leukemia therapy. © 2017 Elsevier Ltd. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. GSK3 and cancer e the early years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. GSK3 and stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. GSK3 as a master regulator of protein turnover involving key components of cell proliferation, cell survival, networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. GSK3 and leukemia microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. GSK3 and leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. GSK3 and leukemia drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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E-mail address: [email protected]. http://dx.doi.org/10.1016/j.jbior.2017.05.001 2212-4926/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ruvolo, P.P., GSK-3 as a novel prognostic indicator in leukemia, Advances in Biological Regulation (2017), http://dx.doi.org/10.1016/j.jbior.2017.05.001

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1. Introduction Aberrant signal transduction to promote tumorigenesis, drug resistance, and metastasis is a common feature of many cancers including leukemia (Kornblau et al., 2006a, 2006b; Davis et al., 2014; Follo et al., 2015; McCubrey et al., 2015, 2016; Mundi et al., 2016; Ruvolo, 2016; McCubrey et al., 2017; Papke and Der, 2017). In acute myeloid leukemia (AML) the activation of survival kinases such as Protein Kinase B (AKT), Protein Kinase C (PKC), and Extracellular Receptor kinase (ERK) has been shown to predict poor clinical outcome for patients with AML (Kornblau et al., 2006a). Dysregulation of the phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) cascade has been found to be central to tumor homeostasis in many cancers (Toker, 2012; Toker and Marmiroli, 2014; Baer et al., 2015; Follo et al., 2015; Jhanwar-Uniyal et al., 2015; McCubrey et al., 2015, 2016, 2017). AKT is central to regulation of gene and protein expression, cell growth, cell survival, and immunogenicity so the kinase has been considered a prime candidate to target for AML therapy (Konopleva et al., 2014; Wang et al., 2016; Brenner et al., 2016). As AKT is a promiscuous kinase with over a thousand confirmed substrates, an understanding of how AKT regulates leukemia cell survival is essential to any strategies to develop anti-AKT drugs. A wellstudied AKT target is Glycogen Synthase Kinase 3 (GSK3). GSK3 controls many diverse pathways that impact cell metabolism, cell differentiation, cell death processes such as apoptosis and autophagy, and may be central to tumorigenesis in certain cancers (Plyte et al., 1992; Harwood, 2001; Woodgett, 2001; Benoit et al., 2014; McCubrey et al., 2014a, 2014b, 2016; Patel and Woodgett, 2017). While many kinases like AKT require a post-translational event for their activation, GSK3 alpha and GSK3 beta are unique in that they are constitutively active and thus depend on negative regulation by other proteins. There are two functional isoforms of GSK3. Both are highly homologous and appear to have redundant functions but they are encoded on genes on separate chromosomes (reviewed in McCubrey et al., 2014a). The human GSK3 alpha (GSKA) gene is located on chromosome 19 while the gene for GSK3 beta (GSK3B) is located on chromosome 3. Based on analysis of GSK3 consensus sequences, it is estimated that GSK3 has more target proteins than any other kinase (Xu et al., 2009). While many GSK3 substrates are involved in drug resistance and tumorigenesis, there is an intense focus on GSK3 as a beta Catenin kinase and its role in WNT signaling (Benoit et al., 2014; McCubrey et al., 2014a, 2014b, 2016, 2017). GSK3 phosphorylation of betacatenin targets the molecule for proteolysis. Failure to control beta-catenin expression can result in expression of pro-tumor molecules such as Cyclin D1, c-MYC, and c-JUN (Xu et al., 2009). GSK3 may also serve as a feedback regulator for survival signaling cascades. Ruzzene et al. (2017) suggest a possible role for GSK3 in Casein Kinase (CK2)/AKT axis by a mechanism involving GSK3 regulation of PTEN. GSK3 is also central to tumor signaling mediated by receptors such as Epidermal Growth Factor Receptor (EGFR) and the RAS oncogene (Fitzgerald et al., 2015). GSK3 is emerging as an important prognostic factor in leukemia though whether active kinase is favorable or unfavorable for outcome depends on the type of leukemia and the GSK target molecules involved. Functional and structural review of GSK will be covered by other investigators in this Special Issue and have been covered elsewhere (Woodgett, 2001; Benoit et al., 2014; McCubrey et al., 2014a, 2016, 2017). In this review, I will cover the role of GSK3 in leukemia cell biology both as tumor suppressor and potential oncogene. We will also cover the various therapeutic approaches that either target GSK3 or rely on GSK3 activation for efficacy. 2. GSK3 and cancer e the early years In the early 1990s, GSK3 was emerging as an important regulator of many cellular functions that are essential for cancer cell homeostasis (Plyte et al., 1992). The diversity of substrates the kinase targeted made it difficult to assess potential antitumor or pro-oncogene role the kinase may play as targeted proteins could be involved in metabolism (e.g. Glycogen Synthase), gene expression (e.g. JUN and MYC), and signal transduction (e.g. the PP2A inhibitor I2, Focal Adhesion Kinase) (Plyte et al., 1992; McCubrey et al., 2014a, 2016, 2014b; Patel and Woodgett, 2017). At the time a role for GSK3 in stem cell biology and differentiation was unknown; however, a Drosophila gene (zeste-white 3) that acts as a critical regulator of embryogenesis was found to be highly homologous with mammalian GSK3 (Plyte et al., 1992; Patel and Woodgett, 2017). Later studies would identify critical roles for GSK3, particularly as a regulator of WNT signaling, in stem cell maintenance and self-renewal and in cell differentiation involving many cell types (reviewed in Sineva and Pospelov, 2014; McCubrey et al., 2014a, 2016; Patel and Woodgett, 2017). T Cell leukemia 1 (TCL1) is a proto-oncogene associated with T cell prolymphocytic leukemia and was shown to act via a mechanism involving activation of AKT (Laine et al., 2000). In 2003, Cheong et al. (2003) examined the prognostic potential of Phosphatase and Tensin homologue (PTEN) phosphorylation in AML. Though phosphorylation of PTEN stabilizes the enzyme, the modification reduces PTEN association with its substrate Phosphatidylinositol 3-Kinase (PI3K) resulting in activation of AKT. In AML patients with elevated PTEN phosphorylation, S21/S9 phosphorylation of GSK3 was found in all cases (Cheong et al., 2003). Phosphorylation of PTEN was shown to be prognostic for poor survival outcome suggesting that inactivation of GSK3 was associated with leukemia cell survival (Cheong et al., 2003). GSK3 has been shown to form a complex with beta Catenin, Adenomatous Polyposis Coli (APC), Axin, and other proteins that served functionally to promote degradation of beta Catenin in the cytosol (Rubinfeld et al., 1996; Aberle et al., 1997; Sineva and Pospelov, 2014). With the knowledge that beta Catenin was a key regulator of stem cells and a common consensus was that cancer stem cells were the Holy Grail for therapy, interest in GSK3 role in cancer stem cell biology grew. The Bhatia laboratory performed an elegant study where they demonstrate loss of GSK3 results in AML (Guezguez et al., 2016). In that study, WNT/beta Catenin was a major pathway upregulated in cells transformed with loss of GSK3 beta. Please cite this article in press as: Ruvolo, P.P., GSK-3 as a novel prognostic indicator in leukemia, Advances in Biological Regulation (2017), http://dx.doi.org/10.1016/j.jbior.2017.05.001

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3. GSK3 and stem cells GSK3 has been shown to regulate gene expression by a diverse range of mechanisms involving control of transcription factors, splicing and RNA processing factors, and expression of non-coding RNA such as microRNAs (Plyte et al., 1992; McCubrey et al., 2014a, 2016; Patel and Woodgett, 2017). The oncogene protein c-MYeloBlastosis (c-MYB) is a key regulator of hematopoiesis and dysregulation of MYB is associated with both lymphoid and myeloid leukemias (reviewed in Zhou and Ness, 2011; Uttarkar et al., 2017). GSK3 beta has been shown to suppress expression of c-MYB in leukemia cell lines including K562 (CML) and Jurkat (T cell ALL; Zhou et al., 2011). The active kinase promotes degradation of the transcription factor. In this instance GSK3 beta actually serves as a pro-survival factor as suppression of MYB results in apoptosis due to loss of expression of BCL2 and Survivin (Zhou et al., 2011). This result was consistent with prior study showing that inhibition of GSK3 while supportive of hematopoiesis actually was deleterious for leukemia cell growth (Holmes et al., 2008). In that study Survivin was also implicated as a key component of the mechanism. GSK3-mediated regulation of gene expression appears to be a critical control component for stem cell pluripotency as GSK3 suppression of beta catenin inhibits transcription of stem cell factors such as Nanog and OCT4 (Marks and Stunnenberg, 2014). Pharmacologic inhibition was shown to induce WNT signaling in human ESC and suppression of GSK3 resulted in maintenance of the undifferentiated state (Sato et al., 2004). Nanog expression was linked to active PI3K/AKT signaling with suppression of GSK3 as being a critical component for expression of the stem cell factor (Storm et al., 2007). Further studies on GSK3 role in stem cell biology revealed that Transcription Factor 3 (TCF3) was central to the GSK3/beta catenin axis in regulating stem cell self-renewal (Yi et al., 2011). Self-renewal of pluripotent stem cells is achieved with a “two hit inhibitor” approach where inhibition of ERK blocks differentiation and blockade of GSK3 supports WNT/beta catenin axis (Sato et al., 2004; Ying et al., 2008; Yi et al., 2011; Marks and Stunnenberg, 2014). The mechanistic role for GSK3 suppression in stem cell selfrenewal clearly involved beta catenin but specific components were not clear. On the one hand, OCT-4 may be the necessary molecule involved or TCF3 (Yi et al., 2011). Ablation of TCF3 was shown to bypass the need for GSK3 inhibition to maintain stem cell self-renewal indicating that at least part of the mechanism involved suppression of TCF3 by active WNT signaling (Yi et al., 2011). In addition, Transcription Factor 1 (TCF1) was shown to be activated by WNT signaling demonstrating that the two TCF members had opposing functions (Yi et al., 2011). Possible other roles for GSK3 in cancer stem cells may involve regulation of PTEN as PTEN has been shown to be critical in leukemia stem cells and other tumor stem cells (Ciuffreda et al., 2014; Fragoso and Barata, 2014). 4. GSK3 as a master regulator of protein turnover involving key components of cell proliferation, cell survival, and cell differentiation networks Whereas beta Catenin was recognized early as a critical molecule in GSK3 mediated pathways essential to cancer cell survival, a large number of new GSK3 targets emerged that were inolictaed in WNT signaling (e.g. Cyclin D1) and also in other pathways such as anti-apoptotic signaling (e.g. MCL-1). Dissection of the WNT pathway identified Cyclin D1 as a protein subject to WNT regulation and the regulatory mechanism involved GSK3 mediated proteolysis of the cell cycle regulator (Diehl et al., 1998; Rimerman et al., 2000; Xu et al., 2009). The critical GSK3 phosphorylation site in Cyclin D1 was found to be Threonine 156 (Diehl et al., 1998). Overexpression of WNT in NIH3T3 cells suppressed GSK activity and increased levels of Cyclin D1 that resulted in accelerated cell proliferation with greater entry of cells into S phase (Rimerman et al., 2000). The Rimerman study (2000) established cross talk between WNT and cell cycle with WNT not only stabilizing the cell cycle regulator but also induced its gene expression as Cyclin D1 is subject to transcriptional regulation by beta Catenin via TCFLEF1. Other WNT regulatory molecules subject to proteolytic regulation by GSK3 include SNAIL (Zhou et al., 2004; Yook et al., 2005) and SMAD1 (Fuentealba et al., 2007). Wei et al. (2005) identified c-JUN as another oncogene that was subject to proteolytic regulation of GSK3. That study also found that MYC and c-JUN could be coordinately regulated by GSK3 and a common E3 ubiquitin ligase (i.e. FBW7). A critical survival regulator of leukemia cells, particularly AML cells, is MCL-1 (Kaufmann et al., 1998; Opferman, 2006; Konopleva et al., 2012; Glaser et al., 2012; Rahmani et al., 2013; Pan et al., 2015). Maurer and colleagues found that IL-3 deprivation resulted in proteolysis of MCL-1 by a GSK3 mediated mechanism (Maurer et al., 2006; Letai, 2006). In the study by Maurer and colleagues, IL-3 withdrawal was shown to reduce MCL-1 protein levels in association with apoptotic cell death. The mechanism of IL-3 withdrawal mediated loss of MCL-1was determined to involve suppression of PI3K/AKT axis resulting in activation of GSK3. Activation of GSK3 in the IL-3 deprived cells led to S159 phosphorylation of MCL-1 with subsequent proteolysis mediated by ubiquitination and proteasomal degradation of the anti-apoptotic molecule. The role of GSK3 was deemed to be essential as inhibition of GSK3 by pharmacological inhibitors (i.e. CHIR-611 and CHIR-911) or by suppression of both alpha and beta isoforms of GSK3 by siRNA prevented MCL-1 degradation and protected cells from death upon IL-3 deprivation (Maurer et al., 2006). Protein half-life studies confirmed that GSK3 phosphorylation of MCL-1 targeted it for destruction. As MCL-1 has an extremely short half-life (less than 4 h in most cells), it is not surprising that stabilizing this protein would favor leukemogenesis and drug resistance (Inuzuka et al., 2011a,b; Bose and Grant, 2013). That a wide variety of oncogenic proteins including MCL-1, MYC, c-JUN, and beta Catenin are negatively regulated by GSK3 emphasizes the critical role the kinase as a tumor suppressor at least in some cell types. A model showing a few of the GSK3 targets and their associated E3 ubiquitin ligases is shown in Fig. 1.

Please cite this article in press as: Ruvolo, P.P., GSK-3 as a novel prognostic indicator in leukemia, Advances in Biological Regulation (2017), http://dx.doi.org/10.1016/j.jbior.2017.05.001

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Fig. 1. Scheme of GSK3 mediated regulation of degradation of key oncogenes. GSK3 phosphorylates MCL-1, beat Catenin, Cyclin D1, and c-MYC at the indicated sites resulting in ubiquitination by an E3 ligase followed by proteasomal mediated destruction.

5. GSK3 and leukemia microenvironment The bone marrow is a critical niche for leukemia cell survival and serves to protect the cancer cells from chemotherapy and immune surveillance (Tabe and Konopleva, 2015; Brenner et al., 2016; Hoggatt et al., 2016; Medyouf, 2017). Support cells in the leukemia microenvironment can either provide factors (e.g. cytokines) that promote survival signaling or can induce survival signaling cascades in response to cell:cell contact. The leukemia cell can also influence support cells. In CLL, microvesicles from leukemia cells introduce cell membrane components such as AXL which activate the AKT pathway and suppress GSK3 activity (Ghosh et al., 2010). At present very little is known about the GSK3 role in the leukemia niche. Mesenchymal stromal cells (MSC) are pluripotent stem cells that give rise to osteoblasts, chondrocytes, and adipocytes (Prockop, 1997; Dennis and Charbord, 2002; Pleyer et al., 2016). Aberrant osteoblast lineage cells have been implicated as drivers of hematologic malignancies by the Scadden and Kousteni groups (Raaijmakers et al., 2010; Kode et al., 2014). Kode et al. (2014) demonstrated that dysregulation of beta Catenin in osteoblasts could promote leukemogenesis. It is not implausible that suppression of GSK3 might be important to allow beta Catenin to function in this scenario. GSK3 has been shown to be a critical regulator of osteoblast differentiation (Seo et al., 2011; D'Alimonte et al., 2013; Biver et al., 2014; Boonanantanasarn et al., 2015; Kumar et al., 2015; Xu et al., 2015). GSK3 also plays a key role in osteoblast cell proliferation by regulating MYC (Smith et al., 2002). Thus GSK3 can impact the support cells in the leukemia niche by at least controlling components derived from MSC. 6. GSK3 and leukemia GSK3 regulates a diverse number of pathways that can impact leukemogensis, leukemia cell survival, potential chemotherapy resistance, and importantly impact leukemia stem cells (LSC). The Jamieson laboratory has linked GSK3 to regulation of LSC and has suggested the kinase plays a key role in progression of chronic myeloid leukemia (CML) cells from chronic phase to blast crisis (Abrahamsson et al., 2009). In that study, a survey of beta catenin targets identified reduced levels of GSK3B but not GSK3A mRNA and protein in blast crisis CML cells. In addition, sequence analysis of cDNA from blast crisis samples from CML patients determined that a misspliced variant of GSK3B (mGSK3B) existed where exons 8 and 9 were deleted. The mGSK3B isoform lacked the ability to bind Axin and FRAT and thus the variant kinase was ineffective in phosphorylating beta-catenin suggesting a key role for this mutant kinase in CML biology (Abrahamsson et al., 2009). Supporting this model, introduction of mGSK3B into chronic phase CML cells increased beta catenin expression and rendered the cells more similar to blast crisis CML cells when studied in in vivo models (Abrahamsson et al., 2009). On the other hand, introducing WT GSK3 into blast crisis CML cells was shown to reduce beta catenin and these cells displayed reduced leukemia engraftment in the mouse model studies. While slice variants of GSK3 in cancers other than CML have not been reported, splice variants of GSK3B have been found in neurological pathologies such as Parkinson ’s disease and bipolar disorders (Kwok et al., 2005; Lin et al., 2013). The presence of mGSK3B in particular CML subpopulations such that a RNA splicing regulatory mechanism is altered at some point during differentiation of the CML cells and in progression from chronic phase to blast crisis. Indeed, the Jamieson laboratory later identified changes in the p150 isoform of Adenosine Deaminase Acting on RNA 1 (ADAR1) expression that might be responsible for production of mGSK3B (Jiang et al., 2013). ADAR1 is a RNA editing enzyme that is regulated by interferon gamma and has been implicated in other cancer other than CML (reviewed in Zipeto et al., 2015). GSK3 has also been implicated in regulation of autophagy mediated survival in CML (Shao et al., 2014). Autophagy has been implicated as a possible mechanism for resistance to imatinib or other tyrosine kinase inhibitors (TKIs) in CML cells including leukemic stem cells (Carew et al., 2007; Bellodi et al., 2009; Drullion et al., 2012). Spautin-1 is a Class III PI3K Please cite this article in press as: Ruvolo, P.P., GSK-3 as a novel prognostic indicator in leukemia, Advances in Biological Regulation (2017), http://dx.doi.org/10.1016/j.jbior.2017.05.001

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inhibitor that has been shown to block autophagy by preventing deubiquitination of Beclin 1 (Liu et al., 2016). Spautin-1 treatment of CML cells enhanced cell killing by a mechanism that involved suppression of AKT and activation of GSK3resulting in MCL-1 degradation (Shao et al., 2014). GSK3 also has been shown BY Yang and colleagues to protect cells from autophagic death by modulating BAX Interacting Factor 1 (BIF1) expression (Yang et al., 2010). In that study, blocking GSK3 was shown to result in switch from autophagy to necrosis or apoptosis during serum starvation by mechanisms involving BIF1. A Korean study of AML patients determined that phosphorylation of Phosphatase and Tensin homologue (PTEN) was associated with shorter survival (Cheong et al., 2003). AML patients with p-PTEN (73.8%) had a reduced probability of survival after 1 year compared to patients with unphosphorylated PTEN (i.e. 38% versus 69%, respectively). Phosphorylation of AKT was observed in >95% of patients and phosphorylation of GSK3 in 100% of patients with p-PTEN while phosphorylation of either of these kinases was not seen in patients with unphosphorylated PTEN (Cheong et al., 2003). While this study did not determine the impact of AKT or GSK3 in the studied patient population, the results did suggest that the presumed suppression of PTEN function could be influencing AML cell survival by activating AKT with resulting suppression of GSK3 function (Cheong et al., 2003). Schwable et al. (2005) determined that Regulator of G-Protein Signaling 2 (RGS2) was suppressed by FLT3 ITD in AML cells with that mutation. RGS2 is a GTPase activating protein that attenuates receptor signaling mediated by G proteins and has been shown to have a role in hematopoiesis (Hepler, 1999; Louwette et al., 2012). Introduction of FLT3 ITD into murine IL3 dependent 32D cells resulted in suppression of RGS2 with activation of AKT and strong phosphorylation of GSK3 (Schwable et al., 2005). Treatment with IL3 but not FLT3 ligand augmented GSK phosphorylation. Interestingly, 32D cells with exogenous WT FLT3 did not display phosphorylation of AKT or GSK3 even when FLT3 was provided but phosphorylation of both kinases was observed with IL3 (Schwable et al., 2005). Introduction of RGS2 to cells with FLT3 ITD reversed phosphorylation of AKT and GSK3 indicating that RGS2 was a negative regulator of AKT/GSK3 axis in FLT3 ITD cells. RGS2 expression was induced in various cell line models of differentiation including HL60 granulocytic differentiation with TPA, NB4 granulocytic differentiation with ATRA, and U937 monocytic differentiation with ATRA and TPA (Schwable et al., 2005). Introduction of FLT3 ITD in NB4 cells blocked differentiation as well as RGS2 induction (Schwable et al., 2005). It was not clear in that study if GSK3 activation was required or necessary for RGS2 mediated differentiation of the various AML cells but GSK3 is well established as a regulator of myeloid differentiation (Gupta et al., 2012; Banerji et al., 2012; Lochab et al., 2013; Hu et al., 2016). Yi et al. (2016) recently demonstrated Integrin beta 3 (ITGB3) expression is prognostic for poor outcome in cytogenetic normal AML especially in patients with FLT3 ITD mutation. Other studies have looked at GSK prognostic potential in AML. Proteomic analysis using reverse phase protein analysis (RPPA) was used to assess phosphorylation status and expression of total GSK3 protein in 511 AML patients (Ruvolo et al., 2015). Patients with higher levels of phosphorylated GSK3 displayed shorter overall survival (OS) compared to those with low levels of phospho-kinase in the total AML population (40 vs 54weeks, respectively; p ¼ 0.015) as well as in the patients with Intermediate Cytogenetics (56 weeks for high expressors vs 77.8 weeks for low expressors; p ¼ 0.018; Ruvolo et al., 2015). Elevated phospho-GSK3 kinase was associated with shorter remission duration in these patient populations. In all AML patients remission duration was significantly shorter in patients with higher levels of phosphorylated kinase compared to those with low levels (43 weeks vs 67 weeks respectively; p ¼ 0.022; Ruvolo et al., 2015). This also held true in the Intermediate Cytogenetic population (45 weeks vs 95 weeks; p ¼ 0.0075) and interestingly, this association held in Intermediate Cytogenetic patients with FLT3 mutations (24 weeks vs 50 weeks respectively; p ¼ 0.009; Ruvolo et al., 2015). AML patients with FLT3 mutations represent roughly one-third of AML patients and as these patients have very poor survival prospects they represent a significant at risk population (Gilliland and Griffin, 2002; Small, 2006; Grunwald and Levis, 2015; Daver et al., 2015). The observation that inactivation of GSK3 influences prognosis in FLT3 mutant patients suggests that inactivation of GSK3 may be critical. The studies by Scwable et al. on RGS2 (2005) and Yi and colleagues on integrin (2016) would support this premise. While GSK3 phosphorylation was significant in the total and Intermediate Cytogenetic AML populations, phosphorylation status of the kinase did not influence OS status in patients with unfavorable cytogenetics or favorable cytogenetics (Ruvolo et al., 2015). The AML study using RPPA also allowed for expression of S21/S9 phospho-GSK3 to be correlated with expression of over 200 other proteins (Ruvolo et al., 2015). As would be expected expression of beta catenin in the AML cells was correlated with GSK3 phosphorylation status suggesting that GSK3 regulation of WNT signaling is important in AML. Interestingly, MCL-1 expression was not correlated with S21/S9 phosphorylation of GSK3 by RPPA. FOXO3A was negatively correlated with phospho-GSK3 though the association was likely due to active AKT as determined by observed effects of pharmacologic inhibition of AKT and GSK on FOXO3A expression (Ruvolo et al., 2015). The RPPA study also identified a positive correlation between Protein Kinase C delta (PKC delta) activation and GSK3 S21/S9 phosphorylation. The prospect of PKC delta as a GSK3 kinase in AML was interesting as Kinehara and colleagues implicated GSK3 inactivation by PKC delta as a mechanism regulating self-renewal in pluripotent stem cells (Kinehara et al., 2013). However, the association between GSK3 and PKC delta appeared to be indirect as AKT was implicated as a PKC delta kinase (Ruvolo et al., 2015). The various associations identified between GSK3 and many other protein representing diverse signaling networks underscores the complexity of GSK3 mechanistic role in AML biology. El-Gamal et al. (2014) found PKC inhibitor AEB071 was effective as an anti-CLL agent using in vitro and in vivo models. AEB071 was found to suppress beta Catenin expression with concomitant suppression of downstream transcriptional targets such as c-Myc and CD44. The mechanism of beta Catenin suppression is not dependent on PKC but rather involves GSK3 (ElPlease cite this article in press as: Ruvolo, P.P., GSK-3 as a novel prognostic indicator in leukemia, Advances in Biological Regulation (2017), http://dx.doi.org/10.1016/j.jbior.2017.05.001

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Gamal et al., 2014). AEB071 activates PP2A to dephosphorylate GSK3 at S21/S9 resulting in activation of the kinase. These results suggest that GSK3 is inactivated in CLL perhaps due to insufficient PP2A activity but activation of the kinase had a potent anti-leukemia effect (El-Gamal et al., 2014). GSK3 inactivation in CLL may also be essential for support of MCL-1 expression. Tyrosine receptor kinase SYK induces PKC delta to phosphorylate and inactivate GSK3 in CLL (Baudot et al., 2009). Pharmacologic inhibition of SYK using BAY61-3606 or suppression of SYK with siRNA induced apoptosis of CLL cells. SYK mediated survival pathway involves protection of MCL-1 from proteasomal degradation as cells could be protected from SYK inhibition by inhibition of GSK3 or by introducing a MCL-1 S159A mutant (Baudot et al., 2009). The implication of PKC delta as the agent that suppressed GSK3 in the CLL cells suggests novel survival function for the PKC isoform and is consistent with RPPA data in AML showing strong correlation between GSK3 S21/S9 phosphorylation and phosphorylation of PKC delta at multiple sites associated with activation (Baudot et al., 2009; Ruvolo et al., 2015). The study by Guezguez et al. (2016) demonstrates the critical importance of GSK3 dysregulation in blood stem cells leading to malignancy. In that study, though allelic deletion of GSK3 alpha had no effect, deletion of GSK3 beta led to a myelodysplastic syndrome (MDS). Hematopoiesis was impaired with a skew toward myeloid differentiation. AML resulted when both GSK3 alpha and GSK3 beta were deleted (Guezguez et al., 2016). Effects on WNT/beta Catenin signaling suggested disruption of GSK3 could influence LSC. A very important finding was that transcriptome profile derived from the animal study (i.e. comparisons of gene expression in WT versus GSK3 beta knockout mice) could be used to predict progression of MDS to AML in both pediatric and adult patients (Guezguez et al., 2016). Patients with reduced expression of transcriptome associated GSK3 beta knockout were significantly more likely to transform to AML and have a poorer survival outcome. Interestingly, the study demonstrated that GSK3 alpha and GSK3 beta had different roles in mDS as only deletion of GSK3 beta resulted in MDS (Guezguez et al., 2016). 7. GSK3 and leukemia drugs In general the thought has been that GSK3 supports pro-survival molecules like beta Catenin, MCL-1, and c-MYC so strategies to activate the kinase would seem the most logical. However, GSK can also mediate pro-survival mechanisms and thus strategies to target GSK for therapy will depend on the particular cell type. A model depicting a few of the pathways where GSK3 could act as a tumor suppressor or tumor promoter are depicted in Fig. 2. As an example where GSK3 serves a tumor suppressor role, AEB071 efficacy as a drug targeting CLL relied on GSK3 activation (El-Gamal et al., 2014). A number of therapeutic strategies to treat leukemia have been shown to involve mechanisms that involve activation of GSK3 often by suppression of PI3K/AKT pathway (reviewed in McCubrey et al., 2014a; McCubrey et al., 2014b). The Grant laboratory observed this phenomenon in their combination of farnesyltranserase inhibitor with CHK1 inhibitor UCN-01 (Dai et al., 2005). The Martelli group found that AKT inhibitor A443654 was potent to induce apoptosis in a variety of T-ALL cells and that the mechanism of killing was at least dependent in part on activation of GSK3 (Fala et al., 2008). An interesting finding from that group was that ds RNA Dependent Protein Kinase (PKR) served a pro-survival role in the ALL cells by suppressing GSK3 (Blalock et al., 2009). The mechanism involves regulation of Protein Phophatase 2A (PP2A) rather than PKR target eIF2 alpha. Endoplasmic reticulum (ER) stress mediated death pathway appears to require GSK3. While AML derived U937 and HL60 cells are sensitive to tunicamycin, BCR-ABL þ CML derived K562 cells are resistant to the ER stress inducing agent unless GSK3 is activated in those cells (Huang et al., 2009). The multi-kinase inhibitor ABT-869 whose targets include FLT3 also operates via a mechanism where AKT is suppressed and GSK3 activated (Hernandez-Davies et al., 2011). Rahmani et al. (2013) demonstrated that use of PI3K inhibitor (BEZ235) or AKT inhibitor (MK2206) with BH3 mimetic against BCL2 and BCL-XL (ABT-737) was

Fig. 2. Scheme of GSK3 pathways that result in pro-tumor or anti-tumor activity. GSK3 phosphorylation of MCL-1, beat Catenin, and c-MYC has tumor suppressor effect as active GSK3 destroys these oncogenic proteins. GSK3 also can activate p53. On the other hand, GSK3 destruction of MYB results in elevated BCL2 and Survivin which promotes tumor survival.

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efficacious against AML cell lines and primary cells and the mechanism was dependent on GSK3. Use of GSK3 pharmacologic inhibitor or GSK3 genetic knock down prevented MCL-1 reduction and thus MCL-1 appears to be a critical target of GSK activation (Rahmani et al., 2013). A number of studies have also emerged where GSK3 was acting to support cancer cells and suggested that suppression of the kinase could have therapeutic benefits in these cases. Wang et al. (2008) found that GSK3 was necessary to maintain leukemia cells with rearrangements of the Mixed Lineage Leukemia (MLL) oncogene. GSK3 mediated degradation of the Cyclin Dependent Kinase Inhibitor (CDKI) p27Kip1 was necessary for cell proliferation and survival of the MLL cells (Wang et al., 2008; Patel and Woodgett, 2008). MLL has been shown to induce p27Kip1 so it is plausible in these cells that GSK3 prevents detrimental effects from expression of the CDKI (Wang et al., 2008). In MLL cells, GSK3 also was found to regulate gene expression of Homeobox genes (HOX) by a mechanism involving phosphorylation of the CREB transcription factor (Wang et al., 2010). GSK3 support of CREB binding with MEIS1 supported gene expression that favored stem cell properties of the MLL cells. Interestingly, suppression of beta Catenin in MLL cells restores sensitivity to GSK3 inhibitors (Yeung et al., 2010). This finding strongly suggests that GSK3 has differential function s in the MLL cells and likely do not act directly in the regulation the beta Catenin in those cells. Another example of GSK3 as a tumor promoter in leukemia involves GSK3 role in daunorubicin resistance in AML cells (De Toni et al., 2006). The mechanism is tumor microenvironment driven with leukemia cell adhesion to osteoblasts driving expression of secreted Frizzled Related Protein (sFRP) antagonizes WNT but activates NF kappa B signaling. A subsequent study found that cell adhesion activated PP2A dephosphorylation of GSK3 though dephosphorylation of the kinase was dependent on involvement of particular integrins (De Toni-Costes et al., 2010). These examples where GSK3 serves as a tumor promoter supported the development of GSK3 inhibitors as therapeutic agents for leukemia. Initial pre-clinical tests of GSK3 inhibitors against cells from diverse leukemia including AML, ALL, and CML were promising (Song et al., 2010). The use of 6-bromoindirubin-30 oxime (BIO) was efficacious when used in in vitro and in vivo models of leukemia. An interesting finding as that co-culture with murine MSC cell line MS5 protected cells from BIO suggesting a complex role for the microenvironment in GSK3 pathways (Song et al., 2010). Another GSK3 inhibitor PDA-66 showed promise in pre-clinical studies using ALL cells (Kretzschmar et al., 2014). GS-87, a highly specific inhibitor of GSK3, has bene shown to induce differentiation of AML cells (Hu et al., 2016). An appealing aspect of this agent is that it had no effect on differentiation of normal blood progenitors and as there was limited toxicity the agent should be well tolerated. A Phase II clinical trial with the GSK3 inhibitor LY2090314 has been performed in AML (Rizzieri et al., 2016). Though the agent showed limited benefit as a single agent it was well tolerated by patients and GSK3 inhibition was established in the patients and thus the drug could have potential benefit perhaps in combination with other drugs. In addition, the potential of GSK3 inhibitors as differentiation agents for therapy need to be further explored. 8. Summary GSK3 is a very versatile molecule in leukemia cell biology. Its potential as both a tumor suppressor and tumor promoter suggests that impact on GSK3 activity should be considered in tailoring therapy for the individual patient. 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