New advances in targeting aberrant signaling pathways in T-cell acute lymphoblastic leukemia

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New advances in targeting aberrant signaling pathways in T-cell acute lymphoblastic leukemia Francesca Paganellia,b,c, Annalisa Lonettid, Laura Anselmie, Alberto M. Martellic, Camilla Evangelistia,b, Francesca Chiarinia,b,∗ a

Institute of Molecular Genetics, Luigi Luca Cavalli-Sforza-CNR National Research Council of Italy, Bologna, Italy IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy c Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy d “Giorgio Prodi” Cancer Research Center, University of Bologna, Bologna, Italy e Department of Biomedical, Metabolic, and Neural Sciences, Section of Morphology, Signal Transduction Unit, University of Modena and Reggio Emilia, Modena, Italy b

A R T IC LE I N F O

ABS TRA CT

Keywords: Target therapies Drug-resistance Cell signaling pathways Metabolism Mutations

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disorder characterized by malignant transformation of immature progenitors primed towards T-cell development. Over the past 15 years, advances in the molecular characterization of T-ALL have uncovered oncogenic key drivers and crucial signaling pathways of this disease, opening new chances for the development of novel therapeutic strategies. Currently, T-ALL patients are still treated with aggressive therapies, consisting of high dose multiagent chemotherapy. To minimize and overcome the unfavorable effects of these regimens, it is critical to identify innovative targets and test selective inhibitors of such targets. Major efforts are being made to develop small molecules against deregulated signaling pathways, which sustain T-ALL cell growth, survival, metabolism, and drug-resistance. This review will focus on recent improvements in the understanding of the signaling pathways involved in the pathogenesis of T-ALL and on the challenging opportunities for T-ALL targeted therapies.

1. T-ALL deregulated pathways, a very complex scenario T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 15% of pediatric and 25% of adult ALL. Despite improvements in treatment over the years, approximately 15% of children and 40% of adults fail to respond to chemotherapy or relapse. Therefore, new therapeutic strategies are urgently needed for this neoplasia (Van Vlierberghe and Ferrando, 2012). T-ALL is a genetically heterogeneous disease, caused by the accumulation of molecular alterations, acting in a multistep pathogenic process. Although the use of high-dose multiagent chemotherapy results in a survival advantage, many patients still relapse and eventually experience refractory leukemia, which is associated with a poor likelihood of survival (Cheng et al., 2017; Evangelisti et al., 2018; Gianfelici et al., 2016; Raetz and Teachey, 2016). Nearly 50% of patients show chromosomal translocations which involve TCR (T-cell receptor) genes, in particular the 14q11 (α and δ TCR) or 7q34 (βTCR) regions. TCR has enhancer activity on genes that are at breakpoints in other chromosomes (Dos Santos

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Corresponding author. Institute of Molecular Genetics, Luigi Luca Cavalli-Sforza-CNR National Research Council of Italy, Bologna, Italy. E-mail address: [email protected] (F. Chiarini).

https://doi.org/10.1016/j.jbior.2019.100649 Received 6 August 2019; Received in revised form 24 August 2019; Accepted 3 September 2019 2212-4926/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Francesca Paganelli, et al., Advances in Biological Regulation, https://doi.org/10.1016/j.jbior.2019.100649

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et al., 2019). Genes involved in the translocations include TAL1, TAL2, LYL1, OLIG2, LMO1, LMO2, TLX1, TLX3, NKX2-1, NKX2-2, NKX2-5, HOXA genes, MYC and Myb. In addition, T-ALL may harbor cryptic rearrangements genes of ABL1 (Iacobucci and Mullighan, 2017). Approximately 70% of T-ALL patients show alterations in cell cycle regulators; the most frequent abnormalities are CDKN2A (Cyclin Dependent Kinase Inhibitor 2A) deletions (loss of heterozygosity at 9p). The CDKN2A locus in chromosome 9 contains the p16INK4A and p14ARF tumor suppressor genes (Ferrando, 2018; Van Vlierberghe and Ferrando, 2012). P16INK4A directly blocks cyclin D-CDK4/6 complexes, whereas p14ARF inhibits Mdm2 (Mouse double minute 2 homolog), a negative regulator of p53 (Van Vlierberghe and Ferrando, 2012). It is also known that Notch (neurogenic locus Notch homolog protein) signaling pathway has a central role in T-ALL. The first evidence of aberrant activation of this pathway was the identification of a chromosomal translocation t(7;9)(q34;q34.3) that juxtaposes NOTCH1 gene next to the TCR locus, leading to the expression of a truncated and constitutively active form of Notch1 (Ferrando, 2018). Moreover, activating mutations have been identified in several domains of Notch. These mutations occur in about 60% of T-ALLs and lead to Notch activation by different mechanisms. Notch1 activation is also caused by FBXW7 (F-box and WD repeat domain containing 7) mutations (15% of T-ALLs cases) that prevent proteasomal degradation of activated Notch1. FBXW7 normally induces Notch ubiquitination via the PEST (proline, glutamic acid, serine, threonine-rich) domain (Li and von Boehmer, 2011; Oliveira et al., 2017). Direct targets of activated Notch, with a role in T-cell leukemogenesis, are C-MYC, pre-TCRα and IL-7Rα (interleukin 7 receptor α). Activation of C-MYC by Notch1 stimulates a feed-forward-loop transcriptional regulatory motif that increases leukemic cell growth (Oliveira et al., 2017; Palomero et al., 2006b). c-Myc is an intermediary between Notch1 and mechanistic target of rapamycin complex 1 (mTORC1) (Chan et al., 2007). Additionally, mechanistic target of rapamycin complex 2 (mTORC2) is a critical regulator of leukemia progression in murine Notch1 mutated T-ALL models (Lee et al., 2012), mediating the activation of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) and CCR7 (C–C chemokine receptor type 7), and leading to increased tissue invasion and death in murine T-ALL models (Buonamici et al., 2009). The high occurrence of mutations in the Notch pathway increased the relevance of understanding whether their prevalence could serve to define disease risk groups. However, studies quantifying the association between the presence of these mutations and T-ALL prognostic features, early response to treatments or patient outcome have provided conflicting evidences, both in pediatric and adult patients, likely due to discrepancies in prognosis reflecting differences in the therapeutic protocols employed for T-ALL patients. Indeed, these variable conclusions regarding the prognostic impact of NOTCH pathway mutations may be due to differences in the population studied, treatment regimens, and co-mutations (Sarmento and Barata, 2011). Phosphatidylinositol 3-Kinase (PI3K)/AKT/mTOR is another pathway frequently hyperactivated in T-ALL; it controls survival, proliferation, apoptosis, migration and metabolism. PI3Ks are a family of lipid kinases activated by growth factors, cytokines and other environmental cues. Additionally, Notch can indirectly modulate PI3K/AKT signaling pathway, by upregulating HES1 (Hes Family BHLH Transcription Factor 1), with consequent transcriptional downregulation of PTEN (phosphatase and tensin homolog), a negative regulator of the PI3K/AKT/mTOR axis (Oliveira et al., 2017; Ruzzene et al., 2017) (Fig. 1). Janus kinase JAK/STAT (signal transducer and activator of transcription) pathway is also hyperactivated in T-ALL (Ferrando, 2018). Different ligands, such as interleukin-7 (IL-7), can activate this pathway. The interaction between IL-7 and interleukin-7 receptor (IL-7R) induces JAK1 and JAK3 phosphorylation and consequently the activation of STAT5. STAT5 translocates to the nucleus where it regulates the transcription of several genes involved in cell survival and proliferation, including the anti-apoptotic BCL-2 (B-cell lymphoma 2) (Girardi et al., 2017) (Fig. 1). Aberrant activation of IL-7/IL-7R signaling and downstream targets (PI3K/AKT/mTOR, JAK/STAT, or (Mitogen-activated protein kinase kinase) MEK/(extracellular signal–regulated kinase)/ERK pathways) is a frequent event in T-ALL (Li et al., 2016). Additionally, Notch1 upregulates the transcription of IL-7R. Different studies showed that deregulation of IL-7/IL-7R pathway could play a role in leukemia progression (Gonzalez-Garcia et al., 2009; Silva et al., 2011). Ras (Rat sarcoma)-mediated signaling is also frequently (about 50% of cases) hyperactivated in T-ALL (Mues and Roose, 2017). Ras proteins includes Harvey-Ras (H-Ras), neuroblastoma-Ras (N-Ras) and Kirsten-Ras (K-Ras) (Mues and Roose, 2017). Ras are a family of small GTP-ases that can be activated by different receptors, including receptor tyrosine kinases and cytokine receptors, and can transmit the signal to MEK/ERK and PI3K/AKT pathways, thereby stimulating cell cycle, survival and metabolic changes (Steelman et al., 2011). Somatic mutations in Ras lock the protein in its active form, leading to overactivation of downstream targets (Mues and Roose, 2017; Oliveira et al., 2017). Mutations in K-Ras and N-Ras occur more frequently in ETP (early T precursor)-ALL than in other subtypes and are present in relapsed ALL patients, where they confer steroid resistance (Irving et al., 2014). RasGRP1 (Ras guanyl-releasing protein 1) expression is tightly regulated during T cell development. Its expression is low in double negative (DN) cells, increases significantly in double positive (DP) cells and peaks in single positive (SP) thymocytes to drop again in peripheral T cells. In normal conditions, RasGRP1 is involved in the activation of T cells, through their TCR, during immunological response (Hartzell et al., 2013). When TCR recognizes specific peptide antigens presented on MHC (major histocompatibility complex) molecules by antigen-presenting cells (APCs), it induces the activation of a signal cascade, which leads to membrane recruitment of RasGRP1, finally activating its substrate Ras. Deregulated RasGRP1 is a very common feature in T-ALL, possibly driving aberrant Ras signaling, through IL-7 pathway (Mues and Roose, 2017). Indeed, in physiological settings T-cells do not activate Ras signaling after cytokine stimulation, but they activate RasGRP1, only when TCR is stimulated. In T-ALL cells, the 2

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Fig. 1. Novel therapies targeting signaling pathways in T-ALL. Wnt binds Frizzled receptor or lipoprotein receptor-related protein (LRP) 5/6. This binding, through Dishevelled (Dvl), inhibits the destruction complex composed by the axin scaffold protein, the tumor suppressor adenomatous polyposis coli (APC), and two serine/threonine kinases: glycogen synthase kinase 3 (GSK3) β, and the casein kinase 1 (CK1). This leads to cytoplasmic accumulation of β-catenin and consequently translocation to the nucleus, where it acts on T cell factor (TCF) and lymphoid enhancing factor (LEF). Notch (neurogenic locus Notch homolog protein) signaling is activated by Delta-like protein 4 (DLL4) binding. Notch protein is normally processed by two proteins, that release the intracellular domain of the Notch protein (NICD). FBXW7 (F-box and WD repeat domain containing 7) normally promotes Notch ubiquitination. The interaction between IL-7 (interleukin 7) and IL-7R (interleukin 7 receptor) leads to phosphorylation of Janus kinase 1 and 3 (JAK1 and JAK3) and activation of signal transducer and activation of transcription (STAT5) proteins. STAT5 dimerizes and moves to the nucleus where acts on different genes, including BCL2, which encodes for Bcl2 mitochondrial antiapoptotic protein. Receptor tyrosine kinases (RTK) are activated by growth factors. The activation leads to the phosphorylation of phosphatidylinositol-4,5 bisphosphate (PIP2) in phosphatidylinositol-3, 4, 5 trisphosphate (PIP3) by phosphatidylinositol-3 kinase (PI3K). Phosphatase and tensin homolog (PTEN) is responsible for PIP3 dephosphorylation. PIP3 recruits the phosphoinositide-dependent kinase-1 (PDK-1) and the serine/threonine kinase (AKT) to the cell membrane, promoting AKT phosphorylation and activation. AKT activates different downstream targets, including the mechanistic target of rapamycin (mTOR). Several inhibitors, which act at different stages in these pathways, are under therapeutic investigation in T-ALL. Small molecule inhibitors are reported in the red boxes. Black arrows show signal transduction. Red lines show inhibition.

overexpression of RasGRP1 activates Ras after cytokine stimulation and this results in strong Ras downstream targets activation and sustained proliferative effects (Coulthard et al., 2009; Hartzell et al., 2013; Mues and Roose, 2017). Besides, T-ALL intracellular signaling is very complex; recent studies demonstrated that approximately 50% of patients (on 146 pediatric T-ALL patients analysed) show, at least, one mutation in PI3K/AKT, Ras, or JAK/STAT pathways, underlining the relevance of activation of these cascades for leukemic T-cells (Cante-Barrett et al., 2016). To add further complexity, cell-extrinsic microenvironmental factors such as nutrient availability, hypoxia, growth factors, could support tumor growth through activation of specific signaling pathways. The intricated molecular mechanisms include a variety of factors whose actions are interrelated one with the other. Accordingly, another fundamental aspect that has to be considered is represented by metabolic reprogramming in leukemic cells. In fact, its role as a key hallmark of cancer cells and its potential as a therapeutic target started to be widely evaluated, also in the context of T-ALL. 2. Targeting PI3K/AKT pathway in T-ALL The PI3K/AKT pathway is one of the most relevant signaling cascades involved in malignant transformation in T-ALL. Through the activation of its multiple downstream targets, it sustains cell survival and proliferation, and controls metabolism (Barata et al., 2004; Cantley, 2002; Oliveira et al., 2017; Song et al., 2005). PI3Ks, as mentioned above, are a family of lipid kinases activated by growth factors, cytokines and other environmental stimuli. Class IA PI3Ks are the most involved in oncogenesis. They are heterodimers with a regulatory subunit (p85) and a catalytic subunit (p110) and are activated by growth factors through either G protein-coupled receptors or tyrosine kinase receptors (Brazil 3

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et al., 2004). PI3K activation leads to the production of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and this newly generated PIP3 localizes the serine/threonine 3-phosphoinositide-dependent protein kinase (PDK1) and mTORC2, as well as AKT, to the plasma membrane. This co-localization catalyzes the phosphorylation of AKT within the kinase domain at threonine 308, whereas mTORC2 phosphorylates serine 473 in the C-terminus domain. When activated, AKT is able to phosphorylate several targets, as Mdm2, Forkhead box protein O1 (FOXO1), Tuberous Sclerosis Complex 2 (TSC2)/tuberin and Caspase-9, acting with an antiapoptotic influence and a proliferative signal. AKT can also translocate to the nucleus, inhibiting apoptosis via the repression of several downstream targets (e.g. Bcl-2-associated death promoter (Bad), BAX, glycogen synthase kinase 3 beta (GSK-3β)) and activation of others (e.g. NF-κB and mTORC1) (Hermida et al., 2017; Vivanco and Sawyers, 2002). The major contribution to the hyperactivation of PI3K/AKT/mTOR pathway in T-ALL derives from PTEN inactivation or deletion (Bongiovanni et al., 2017; Oliveira et al., 2017; Thorpe et al., 2015). PTEN is a lipid phosphatase that dephosphorylates PIP3 to phosphatidylinositol-4,5 bisphosphate (PIP2), thus regulating the PI3K/AKT/mTOR signaling. PTEN can be inhibited by mutational inactivation, gene deletion or post-translational inactivation (Bongiovanni et al., 2017; Oliveira et al., 2017; Thorpe et al., 2015). Mutations or deletions in PTEN tumor suppressor gene have been described in about 12% of adults and 22% of children affected by T-ALL (Chiaretti et al., 2016). PTEN non-sense or frame-shift mutations in exon 7 lead to the production of a C-terminal truncated protein, which is rapidly degraded; of note, these mutations are associated with a poor prognosis. Additionally, non-genetic mechanisms, such as casein kinase 2 (CK2)-mediated phosphorylation and reactive oxygen species (ROS)-induced oxidation, are able to inactivate PTEN, affecting its lipid phosphatase activity (Gowda et al., 2017; Silva et al., 2008). Furthermore, Notch1 can act upregulating HES1, causing a consequent transcriptional downregulation of PTEN and this contributes to the hyperactivation of PI3K/AKT/mTOR axis (Palomero et al., 2007). It has been shown that PTEN mRNA can be targeted by miR-19 (Mavrakis et al., 2010) or c-Myc (Gutierrez et al., 2011) and that Notch1 could control the dynamic exchanges of regulatory B subunits of protein phosphatase 2A (PP2A), causing a decreased affinity of this phosphatase for critical targets such as phospho-AKT (Hales et al., 2013). Hyperactivation of PI3K/AKT pathway due to gain-of-function mutations in p85 and p110 subunits (4–5% of T-ALL cases) or in AKT (2–3% cases) have also been reported (Bongiovanni et al., 2017). However, genetic alterations are not sufficient to account for the very high frequency of PI3K signaling hyperactivation in T-ALL (Gutierrez et al., 2009; Silva et al., 2008). In fact, IGF2, IGFL4, MKNK2, AKT1 genomic gains were also reported altering the PI3K/AKT cascade (Remke et al., 2009). Contrary to PTEN inactivation, these abnormalities do not correlate with a clinical outcome (Gutierrez et al., 2009). Regarding PI3K, it has been demonstrated that the p110γ and p110δ PI3K catalytic subunits sustain leukemogenesis in PTENmutant mice (Subramaniam et al., 2012). Importantly, recent evidences showed that PTEN also represses PI3K/AKT signaling acting via Ikaros transcription factor/miR-26b axis that directly downregulates the expression of PIK3CD, the gene encoding the p110δ PI3K catalytic subunit (Yuan et al., 2017). Given the widespread constitutive activation of PI3K/AKT/mTOR signaling, this cascade has been evaluated as a potential therapeutic target in T-ALL. Several pan-PI3K class I inhibitors demonstrated strong anti-leukemia activity against T-ALL cell lines and primary patient samples (Lonetti et al., 2014, 2015). NVP-BKM120 induced apoptosis in primary patient samples with hyperactivation of PI3K/AKT/mTOR pathway (Lonetti et al., 2014; Silva et al., 2008). This drug also exhibited in vivo anti leukemic activity, increasing the life-span of subcutaneous xenotransplanted mouse models of human T-ALL and synergized with chemotherapeutic drugs currently employed for treating T-ALL patients (Lonetti et al., 2014). NVP-BKM120 is under investigation for patients with advanced leukemia in a phase I clinical trial (NCT01396499) (Ragon et al., 2017). The pan PI3K inhibitor ZSTK-474, combined with nelarabine, inhibited the growth of T-ALL cells and was synergistic in decreasing cell survival and inducing apoptosis in nelarabine-resistant T-ALL primary samples. The drug combination caused dephosphorylation of AKT and the downregulation of Bcl-2, while nelarabine alone induced an increase in phosphorylated-AKT and Bcl-2 signaling in the resistant T-ALL cells and relapsed patient samples, confirming that the PI3K signaling is linked to chemo-resistance to therapy in T-ALL (Lonetti et al., 2016) and that this combination could be a possible strategy for relapsed patients. In PTEN-null models of T-ALL, the dual p110γ/δ PI3K inhibitor, CAL-130, prolonged survival in an animal models. Moreover, the drug blocked proliferation and was able to activate pro-apoptotic signaling pathways in T-ALL cell lines and primary samples (Subramaniam et al., 2012). Conversely, other groups showed that inhibition of all four isoforms of the p110 PI3K catalytic subunit was more effective than dual p110γ/δ PI3K inhibition in inducing cytotoxicity in human PTEN-null T-ALL cells (Lonetti et al., 2015; Stengel et al., 2013). It is known that the hyperactivation of PI3K/AKT/mTOR signaling can impair glucocorticoid response (Piovan et al., 2013; Wei et al., 2006) and targeting PI3K, AKT, and mTOR can enhance the antileukemic effects of glucocorticoids (Piovan et al., 2013; Subramaniam et al., 2012; Wei et al., 2006). Moreover, rapamycin, an allosteric mTORC1 inhibitor, may modulate glucocorticoid resistance, an important indicator of therapeutic failure in T-ALL (Wei et al., 2006). Recently, it has been demonstrated that MK-2206, an orally active allosteric AKT inhibitor, inhibited PI3K/AKT/mTOR signaling, causing a reduction of phosphorylated mTOR in T-ALL (Cani et al., 2015). Moreover, MK-2206 dephosphorylated the glucocorticoid receptor NR3C1 in position serine134, enabling its translocation into the nucleus and restoring steroid sensitivity in T-ALL glucocorticoid resistant cell lines (Piovan et al., 2013). Moreover, xenograft models of glucocorticoid resistant T-ALL treated with MK-2206 and dexamethasone displayed a significant reduction of tumor burden, once again suggesting that AKT influences glucocorticoid 4

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resistant cells in T-ALL (Piovan et al., 2013). AKT inhibition with MK-2206 also showed promising results in stimulating autophagy and targeting a putative leukemia-initiating cell (LIC) population (Simioni et al., 2012). mTOR inhibition in T-ALL is still under investigation, although recent data implicate mTOR activation in the development of early T progenitors and T-ALL (Schubbert et al., 2014). In a mouse model of T-ALL with K-Ras activation, Raptor deficiency (a crucial element of mTORC1 complex), inhibited cell cycle progression in oncogenic K-Ras-expressing T-cell progenitors, and prevented the development of the disease (Schubbert et al., 2014). On the other hand, rapamycin, a well-known allosteric inhibitor of mTOR, extended cell survival of T-ALL bearing mice, but the exposure for prolonged time induced the appearance of rapamycin-insensitive leukemia cells, leading to disease progression (Schubbert et al., 2014). However, through feed-forward- loops between mTOR, PI3K and AKT, inhibition of mTOR often leads to hyperactivation of PI3K/AKT/mTOR cascade (Schwarzer et al., 2015). This observation drove scientists to explore other possible targets to hit therapeutically PI3/AKT/mTOR pathway (Evangelisti et al., 2018). To this aim, dual PI3K/mTOR inhibitors were developed, firstly to overcome the possible issue of drug resistance caused by individual drug administration and secondly, to overcome issues related to feed-forward-loops. In T-ALL, PI-103 and NVP-BEZ235 showed cytotoxic activity against T-ALL patient samples (Chiarini et al., 2009, 2010). Interestingly, the dual PI3K/mTOR inhibitor PI103 determined upregulation of Notch1 target genes, including C-MYC. Accordingly, the combination of PI-103 with either a γsecretase inhibitor (GSI) (such as L-685) or a c-Myc inhibitor (10058-F4) enhanced the effectiveness of PI-103 (Shepherd et al., 2013). Inhibition of the PI3K/mTOR pathway with PKI-587, another dual PI3K/mTOR inhibitor, delayed tumor progression, reduced tumor load and enhanced the survival in immuno-deficient mouse xenograft models (Gazi et al., 2017). In conclusion, several preclinical data on PI3K/AKT/mTOR signaling inhibition in T-ALL showed promising effects. However, relatively disappointing results of clinical trials were obtained, likely due to the development of chemo-resistance mechanisms. For this reason, more rational strategies of combination therapy are necessary to maximize clinical response and minimize toxicity of these drugs. 3. IL-7R/JAK/STAT targeting in T-ALL IL-7 is a type I cytokine produced by bone marrow and thymic stroma which interacts with the IL-7R composed of the IL-7 specific IL-7Rα chain (CD127) and the common γ-chain (γc or CD132), which is shared with others cytokines (Oliveira et al., 2019; Waickman et al., 2016). JAK1 and JAK3 are constitutively associated with the cytosolic region of IL-7Rα and γc, to transduce IL-7 signaling (Waickman et al., 2016). The binding of IL-7 to its receptor leads to IL7-Rα/γc heterodimerization and JAK1 and JAK3 transactivation, resulting in the phosphorylation of the intracellular domain of IL7-Rα. This leads to the recruitment and phosphorylation of STAT5, which dimerizes and moves to the nucleus where it regulates the transcription of target genes such as BCL-2 family members. IL-7 can also activate the PI3K/AKT/mTOR and MEK/ERK pathways (Bongiovanni et al., 2017; Oliveira et al., 2017). Tlymphocytes do not produce IL-7 depending exclusively on microenvironmental IL-7 (Ribeiro et al., 2013). The IL-7/IL-7R pathway has a crucial role in thymocyte development as the expression of IL-7R is tightly regulated during this process. In fact, IL-7R is expressed at high levels at the DN (CD4− CD8−) stage, decreases at the DP (CD4+ CD8+) stage and it is re-expressed at the SP (CD4+ or CD8+) stage (Akashi et al., 1998; Van De Wiele et al., 2004). In humans, loss-of-function mutations in IL-7Rα or in γc, cause severe combined immune deficiency (SCID) syndrome (Noguchi et al., 1993; Puel et al., 1998), while defective signaling in IL-7−/− or IL7R−/− knockout mice leads to early thymic development arrest and lymphopenia (Peschon et al., 1994; Puel et al., 1998; von Freeden-Jeffry et al., 1995). It has been shown that IL-7 contributes to the progression of human T-cell acute lymphoblastic leukemia (Silva et al., 2011). This cytokine has an oncogenic potential in vivo, as demonstrated by the developing of B- and T-cell lymphomas in IL-7 transgenic mice (Abraham et al., 2005; Rich et al., 1993). IL-7 receptor is expressed both in normal T lymphocytes and T-ALL cells. IL-7 promotes cell cycle progression and survival of TALL cells by downregulating the cyclin-dependent kinase inhibitor p27(kip1) and upregulating Bcl-2 in a PI3K/AKT-dependent manner (Barata et al., 2001, 2004). Instead, in normal T-cells, the activation of PI3K pathway by IL-7 stimulation does not affect Bcl-2 expression, resulting only in cell cycle entry (Rathmell et al., 2001; Swainson et al., 2007). Moreover, IL-7-mediated upregulation of Bcl-2 in T-ALL is independent of STAT5 activity. Indeed, STAT5 downregulates BCL-6 and promotes the expression of PIM1 (ProtoOncogene, Serine/Threonine Kinase) which plays a role in mediating IL-7 survival mechanism in T-ALL cells (Ribeiro et al., 2018). Additionally, Notch1 upregulates the transcription of IL-7Rα, and this mechanism appears to be involved in Notch-mediated leukemia cell maintenance (Gonzalez-Garcia et al., 2009; Weng et al., 2004). Around 10% of T-ALL patients display gain of function mutations in the IL-7R gene, and IL-7R mutations occur in all subtypes of TALL (Liu et al., 2017; Oliveira et al., 2019). All mutations are located in exon 6, and most of them result in an unpaired cysteine in the extracellular transmembrane portion of IL-7Rα which promotes receptor homodimerization through two mutant α chains, leading to constitutive activation of the signaling in ligand-independent manner. Moreover, some non-cysteine mutations do not promote homodimerization, but increase responsiveness to IL-7 (Cante-Barrett et al., 2016; Oliveira et al., 2019). IL-7R mutations are not prognostically relevant at diagnosis, but they are predictive of poor response to treatment in relapsed pediatric T-ALL patients (Richter-Pechanska et al., 2017). Additionally, gain of functions mutations have been described in IL-7R downstream targets, such as JAK/STAT. JAK1 mutations occur in about 5% of pediatric T-ALL patients and in 7% of adult patients, while JAK3 mutations are present in approximately 8% and 12% of pediatric and adult T-ALL cases, respectively (Oliveira et al., 2017). Among STAT genes, mutations occur mainly in STAT5 in T-ALL. These mutations were mapped predominantly to the Src homology 2-(SH2) domain – a site that is involved in docking of 5

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phosphorylated tyrosine residues, transient binding to cytokine receptors and binding to other STAT proteins that mediate dimerization and nuclear localization (Koskela et al., 2012). STAT5b can show an activating mutation (N642H) which leads to growth factor-independent cellular proliferation (Bandapalli et al., 2014) and that is associated with an increased risk of recurrence in T-ALL pediatric patients (Chiaretti et al., 2016). Additionally, deletions or inactivating mutations of the protein phosphatase PTPN2 gene lead to hyperphosphorylation of STAT5 in about 6% of T-ALL adult patients (Kleppe et al., 2010). Interestingly, IL-7R signaling pathway can be hyperactivated in patients who do not show genetic alterations in IL-7R, JAK, or STAT5 genes, suggesting that others mechanisms are involved in the activation of IL-7R cascade (Goossens et al., 2015; Tremblay et al., 2016). Different loss of function mutations have been described in the DNM2 (Dynamin 2) gene, normally involved in clathrin-dependent endocytosis of IL-7R. These mutations cause an increased expression and activity of IL-7R signaling in T-ALL cells (Bongiovanni et al., 2017). Given the high frequency of T-ALL patients (about 70% of the cases) who express IL-7R, targeting the IL-7/IL-7R pathway could be therapeutically exploitable (Oliveira et al., 2019). The use of antioxidants (e.g. N-acetylcysteine) is a therapeutic strategy to disrupt mutant IL-7Rα homodimerization, promoting apoptosis of IL-7Rα mutant cells in vitro, and decreasing leukemia progression in vivo (Mansour et al., 2015). Development of antibodies against IL-7Rα and/or against mutant homodimers would be potentially exploitable. Indeed, the use of a murine anti-IL-7Rα antibody conjugated to a cytotoxic agent was able to overcome glucocorticoid resistance (frequent event at relapse) in T-ALL (Yasunaga et al., 2017). Recently, Barata et al. generated a fully human IgG1 (immunoglobulin G) monoclonal antibody against both wild-type and mutant human IL-7Rα. The antibody downregulated IL-7/IL-7R-mediated signaling, sensitized T-ALL cells to treatment with dexamethasone and induced cell death, thereby promoting mice survival rates (Akkapeddi et al., 2019). Several strategies have been developed for targeting not only IL-7 itself but also its downstream signaling elements. Different STAT inhibitors (C1889, pimozide, S31201, STA21) are now available (Dorritie et al., 2014). Ruxolitinib and tofacinib (which target JAK1, JAK2 and JAK3) have shown promising results (Degryse et al., 2014; Waibel et al., 2013). Ruxolitinib showed therapeutic efficacy in ETP-ALL primary patient xenografts in vivo (Maude et al., 2015). JAK inhibitors induced cell death in IL-7R mutant (Senkevitch et al., 2018; Shochat et al., 2011; Zenatti et al., 2011) and IL-7-dependent cells (Barata et al., 2004; Melao et al., 2016). Mutations in IL-7R or in downstream targets confer glucocorticoid resistance and are linked with poor clinical outcome in pediatric TALL patients (Li et al., 2016). However, the use of inhibitors of IL-7R/JAK/STAT pathway was able to overcome glucocorticoid resistance in human ETP-ALL subsets. Indeed, ruxolitinib sensitized T-ALL cells to dexamethasone-induced apoptosis (Delgado-Martin et al., 2017). Moreover, the combined use of JAK1/3 inhibitor tofacitinib and Bcl-2 inhibitor venetoclax (ABT-199) in JAK3 mutant TALL cells showed a synergistic effect compared to either inhibitor employed as single agent (Degryse et al., 2018). Moreover, IL-7Rmutated mouse T-cells showed similar results for ruxolitinib and venetoclax (Senkevitch et al., 2018). Additionally, a recent study has shown that resistance to single agent chemotherapy could be overcome by combining MEK inhibitors with PI3K/AKT/mTOR inhibitors (Cante-Barrett et al., 2016). These drugs had synergistic cytotoxic effects in leukemic cells of patients with mutations in the IL-7R signaling pathway, blocking the IL-7 mediated cascade in T-ALL cells. These results could be translated in vivo to better assess the therapeutic response. 4. Notch1 targeting in T-ALL As we have already mentioned, one of the main mechanisms for T-cell precursors expansion in the context of T-ALL is represented by Notch signaling deregulation. Even though other Notch isoforms, including Notch2 and Notch3, seem to be able to induce T-cell leukemias when expressed in T-cell progenitors, sporadic mutations are found only in NOTCH1, suggesting its fundamental role in driving this pathology. The most common NOTCH1 mutations (40–45% of tumors) involve exon 26 or 27 which encode the N- and Cterminal halves of the heterodimerization domain (HD), respectively. PEST mutations (20–30% of tumors) are found in exon 34 and consist of point mutations. Approximately, 10–20% of primary T-ALLs have both types of mutations, and they always present them in cis in the same NOTCH1 allele, which produces a synergistic increase in Notch1 signaling. In 15% of T-ALL cases, there are FBXW7 mutations or deletions (Aster and Pear, 2001). In T-cell precursors, Notch1 has been shown to regulate cell growth and survival through activation of PI3K/AKT/mTOR signaling pathway, within HES1-mediated PTEN suppression. Finally, Notch1 can upregulate NF-kB activity, directly controlling the expression of IL-7Rα, and may also regulate p53 activity (Ferrando, 2009). Notch1 key role in leukemogenesis easily highlights its potential importance as a molecular therapeutic target, in particular for chemotherapy refractory patients. GSIs originally designed to inhibit the processing of APP (amyloid precursor protein) in Alzheimer's disease, block Notch signaling in T-ALL (Palomero et al., 2006a). Afterwards, this observation drove the initiation of the Dana-Farber Cancer Institute 04–390 trial, a phase I clinical trial testing the activity of MK-0752, an oral GSI developed by Merck for the treatment of relapsed T-ALL patients, which ended with no responses, while side toxicity effects were high (Deangelo et al., 2006). A phase I trial (I6F-MC-JJCA) investigated the safety and anti-tumor activity of LY3039478, a selective oral Notch inhibitor, in an expansion cohort of patients with adenoid cystic carcinoma (ACC), who received the dose-escalation-recommended phase 2 dose (RP2D), established in a previous phase 1 clinical trial in advanced and metastatic cancers (NCT01695005) (Even et al., 2019; Massard et al., 2018). Other GSI inhibitors phase I-II-tested are BMS-906024, PF-3084014, RO4929097. However, GSI resistance can occur, suggesting that other mechanisms compensate Notch pathway to support leukemic cells growth. Currently, the main problem connected to the use of GSIs is represented by the high levels of gastrointestinal toxicity, as these small molecules are not selective for individual Notch receptors. New GSIs have entered in clinical trials (e.g. PF-3084014 or 6

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BMS-906024) (Papayannidis et al., 2015) but so far, no evidence of therapeutic efficacy has been reported. Developing more specific compounds, which can be evaluated in combination with other therapies to sustain acceptable levels of side effects and limit leukemic cells expansion, is an urgent need (Bellavia et al., 2018). An alternative therapeutic option is exemplified by selective Notch1 receptors inhibitory antibodies. Anti-NRR (negative regulatory region) antibodies, such as OMP-52M51, specifically target the NRR region preventing co-activators of Notch1 from binding and also act as Notch1 antagonists, to an even higher extent if combined with dexamethasone (Agnusdei et al., 2014). Other antibodies are currently in preclinical studies, and these include inhibitors of the extracellular domain of γ-secretases. The complete activation of Notch pathway depends on three proteolytic cleavages (S1–S3), and these can represent therapeutic targets as well. In particular, S1 cleavage takes place in the ER (endoplasmic reticulum)/Golgi apparatus, where Notch receptors are first processed. Ca++ is a limiting factor of this reaction and its concentration is finely regulated by SERCA pumps (Roti et al., 2013), whose inhibition through thapsigargin was shown to inhibit Notch1 signaling in T-ALL. JQ-FT is a new compound in which folate was conjugated to an alcohol derivative of thapsigargin via a cleavable ester linkage. The drug is recognized by folate receptors on the plasma membrane and delivered into leukemia cells. JQ-FT has shown NOTCH1 inhibition in vitro and in vivo in mechanistic and translational models of T-ALL (Roti et al., 2018). Once cleaved, the intracellular domain of Notch (NICD) is subjected to different post-translational modifications, which regulate its function and stability. Preliminary data obtained in a Notch3-induced T-ALL mouse model, indicate that targeting the HDAC (Histone Deacetylase) by using Trichostatin A impairs the acetylation/deacetylation of N3ICD, promoting a regression in tumor development (Palermo et al., 2012). Combination of Notch1 signaling inhibition and inhibition of other specific pathways has currently been evaluated. The use of GSIs together with glucocorticoids reverses glucocorticoid resistance in T-ALL cell lines and prevents side toxicity (Dyczynski et al., 2018). Further possible combinations include the use of GSIs together with conventional chemotherapeutic drugs, inhibitors of PI3K/ AKT/mTOR pathway and of NF-kB signaling. Nevertheless, leukemic cells constantly interact with the surrounding microenvironment, creating a niche in which stromal cells play a key role in supporting tumor cells adhesion and migration (Calvo et al., 2019). By the alteration of these interactions, represented for instance by CXCR4 antagonists (e.g. AMD3100 and BL-8040), it is possible to enhance drug targeting of leukemic cells and possibly open a further therapeutic window to treat Notch1-induced T-ALL (Juarez et al., 2007). 5. Wnt/β-catenin signaling pathway and its potential targeting Wnt/β-catenin signaling pathway is determinant in the early development and in hematopoiesis, where it regulates stem cell maintenance and self-rewanal (Luis et al., 2012). The canonical Wnt/β-catenin pathway is activated by the interaction of Wnt ligands to the seven transmembrane-domain protein receptors Frizzled (Fzd) and/or by the low-density lipoprotein receptor-related protein (LRP) 5/6. This binding inhibits the destruction complex, a multiprotein complex that, in the absence of Wnt, controls the cytoplasmic amount of β-catenin via phosphorylation, targeting β-catenin for proteasomal degradation (Stamos and Weis, 2013). It consists of the axin scaffold protein, the tumor suppressor adenomatous polyposis coli (APC), and two serine/threonine kinases: glycogen synthase kinase 3 (GSK3) β, and casein kinase 1 (CK1). When Wnt binds to Fzd and LRP5/6 receptors, Dishevelled (Dvl) displaces GSK3β from axin degradasome and prevents the phosphorylation of β-catenin, allowing its stabilization and accumulation in the cytoplasm (Stamos et al., 2014). Stabilized β-catenin can translocate to the nucleus, where it forms a complex with transcription factors, notably T cell factor (TCF) and lymphoid enhancing factor (LEF), TCF/LEF, thereby activating target genes involved in cell growth and survival, including C-MYC, CCND1, BIRC5, and CDKN1a (Niehrs, 2012). Recently, it has been reported that upregulation of Wnt/β-catenin axis leads to uncontrolled hematopoietic stem cells (HSCs) selfrenewal, generating leukemia stem cells (LSCs) (Lento et al., 2013). Aberrant activation of Wnt/β-catenin signaling has been associated with hematologic neoplastic disorders. Deregulation of Wnt/ β-catenin pathway has been shown in chronic and acute myeloid leukemia (Buonamici et al., 2009; Wang et al., 2010), and recently it has been observed that altered Wnt/β-catenin cascade is involved in drug-resistance in ALL (Dandekar et al., 2014; Gang et al., 2014). Interestingly, more than 80% of pediatric T-ALL patients displayed high β-catenin expression as well as β-catenin-dependent gene activation, including C-MYC, BIRC5, TCF1, and LEF (Agnusdei et al., 2014). Even though 60% of T-ALL harbors mutations of NOTCH1, it has been demonstrated that β-catenin-dependent c-Myc overexpression drives to ALL (Kaveri et al., 2013). It has been shown that stabilization of β-catenin in developing T-cells results in the accumulation of DP T-cells and consequent development of a Notch-independent leukemia with c-Myc overactivation (Guo et al., 2007); (Guo et al., 2008). It has been also shown that the hyperactivation of Wnt pathway could be a leukemia initiating event comparable to Notch signaling (Ng et al., 2014). Overactivation of Wnt signaling seems to be typical of minor subpopulations of the bulk tumors, and these Wnt-active subsets seem to be highly enriched in leukemia initiating activity. In this context, activated β-catenin and Hif (Hypoxia-inducible factor)-1α sustain LSCs, while genetic alterations in β-catenin or Hif-1α could cause a reduction in stem cell frequency, having a minimal impact on the growth of bulk cancer cells (Giambra et al., 2015). This study identified a functional dependency that can be targeted therapeutically in patients with T-ALL, suggesting that pharmacologic inhibitors may improve clinical outcome, mainly for patients with a refractory/relapsed disease (Giambra et al., 2015). Wnt/β-catenin signaling pathway could also play a controversial role in leukemia development. On one side, upregulation of LEF1 7

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expression activates Wnt/β-catenin cascade, leading to T-ALL cell proliferation (Guo et al., 2015). On the other side, TCF1 acts as a tumor suppressor in T-lymphocytes (Lee et al., 2012). Deletions and mutations in LEF1 have been found in T-ALL (Gutierrez et al., 2011). Moreover, TCF1 mutations could promote leukemogenesis, with an aberrant activation of Wnt signaling in leukemic blasts (Tiemessen et al., 2012). It has been demonstrated that LEF1 is increased in TCF1 knockout mice, determining a T-ALL higher incidence. LEF1 overexpression could be due to a decreased expression of TCF1, or mutations in the LEF1 gene, and mutations in genes of other signaling pathways, whose proteins are involved in the regulation of LEF1 expression in T-ALL (Gekas et al., 2016; Giambra et al., 2015; Gonzalez-Garcia et al., 2009; Guo et al., 2015). Of note, a pivotal role of LEF1 has been detected in mice with leukemia induced by activated forms of Notch that were found to directly transcriptionally control LEF1 expression (Spaulding et al., 2007). Given the importance played by constitutive activation of Wnt/β-catenin signaling pathway in the pathophysiology of leukemia, different Wnt/β-catenin inhibitors might be employed as innovative treatments for T-ALL. Furthermore, treatment with the tankyrase inhibitors (i.e. XAV-939) also led to decreased proliferation in in vitro studies (Giambra et al., 2015). Besides, targeting LSCs is an urgent need for this disease. CWP232291, a Wnt/β-catenin inhibitor that blocks the β-catenin-dependent gene expression, is currently tested in acute myeloid leukemia settings (NCT01398462). Another promising drug is ICG-001, a small molecule that antagonizes specifically the binding of β-catenin with CREB-binding protein (CBP), blocking the β-catenin-dependent transcription (Emami et al., 2004). It has been demonstrated that targeting β-catenin, using specific CBP/catenin antagonists in combination with conventional therapy could represent a promising therapeutic strategy to eradicate drug resistant LSCs. It has also been shown that combining ICG001 with conventional drugs leads to eradication of drug-resistant primary leukemia, regardless of CBP mutational status and chromosomal aberration. Moreover this combination increases the survival of NOD/SCID mice engrafted with primary ALL, representing a novel approach to overcome relapse in ALL (Gang et al., 2014). Besides, a second generation clinical compound PRI-724 is now in clinical trials for hematological malignancies (NCT01606579). 6. Bcl-2 targeting in T-ALL Apoptosis is an evolutionary conserved mechanism of programmed cell death that is crucial for the regulation of many processes including embryogenesis, organ development and tissue homeostasis, but it is deregulated in oncologic diseases (Hotchkiss et al., 2009). Apoptosis is triggered by two major pathways, the extrinsic or death receptor pathway (activated by extracellular ligands) and the intrinsic or mitochondrial pathway (activated by stress signals), with the latter frequently perturbed in lymphoid malignancies. The intrinsic apoptotic pathway is orchestrated by Bcl-2 family proteins consisting of the BH3-only and pro-apoptotic (pro-death) proteins Bad, BIK, NOXA, BMF, PUMA, BIM, BID, and HRK, the multi-BH domain proteins BAX and BAK, and the anti-apoptotic (prosurvival) proteins Bcl-2, Bcl-XL, BCL-W, A1, and MCL-1 (Hotchkiss et al., 2009). Normally, pro-survival proteins inhibit the cell death mediators BAX and BAK thus sustaining cell survival. In the presence of stress signals, the BH3-only proteins bind to and inhibit prosurvival members, resulting in BAX and BAK activation that in turn produces mitochondrial outer membrane permeabilization (MOMP) and consequent apoptosis (Hotchkiss et al., 2009). The balance between the anti-apoptotic and pro-apoptotic Bcl-2 family proteins is crucial in determining cell fate, and alterations in protein regulation/expression, that ultimately culminate in apoptosis impairment, have been described in hematological malignancies, including T-ALL (Follini et al., 2019). Since the 1990s a series of molecules targeting Bcl-2 family proteins started to be developed, and the so called BH3 mimetics, that functionally mimic BH3-only proteins by stabilizing the pro-apoptotic proteins, reached significant achievements (Vogler et al., 2017). BH3 mimetics include ABT737, with specificity against Bcl-XL, Bcl-2, and BCL-W; ABT-263 (Navitoclax), a dual inhibitor of Bcl-2 and Bcl-XL; and ABT-199 (Venetoclax) a selective Bcl-2 inhibitor. ABT-737 demonstrated potent antitumor activity in pre-clinical models; however, it had poor pharmacokinetic properties for clinical development, while ABT-263 induced excessive toxicity during clinical trials, that limited its usefulness in patients (Scheffold et al., 2018). By contrast, ABT-199 displayed a safe profile and it was FDA-approved for the treatment of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia patients (Yalniz and Wierda, 2019). Chonghaile et al. were pioneers in demonstrating the relation between T-ALL maturation stage and the sensitivity to Bcl-2 inhibition (Chonghaile et al., 2014). By using BH3 profiling, these authors demonstrated that T-ALL are often dependent on Bcl-XL, while ETP-ALL, a very high risk for relapse T-ALL subtype, is dependent on Bcl-2, and sensitive to ABT-199 both in vitro and in vivo (Chonghaile et al., 2014). Further preclinical studies corroborated these findings, highlighting high sensitivity to Bcl-2 inhibition, upon ABT-199 treatment, of Loucy cell line, the only T-ALL cell line known to be representative of ETP-ALL, as well as immature primary T-ALL samples (Anderson et al., 2014; Peirs et al., 2014). Indeed, both Loucy and immature T-ALL cells, defined as TLX1-, TLX3-or HOXA-molecular subtypes, displayed high levels of Bcl-2, relative to mature T-ALL cells, often characterized by TAL1 or LMO2 oncogene activation, which poorly responded to ABT-199 treatment. In addition, ABT-199 was synergistic in combination with chemotherapeutics agents including cytarabine, doxorubicin, l-asparaginase, and dexamethasone (Anderson et al., 2014; Peirs et al., 2014). In T-ALL, Bcl-2 over-expression is the result of aberrantly activated signaling pathways, including activated oncogenic JAK2 that in turn regulates the key survival PI3K/AKT/mTOR and MEK/ ERK pathways (Oliveira et al., 2019). However, at least in T-ALL driven by TEL-JAK2 fusion, targeting PI3K/AKT/mTOR or MEK/ERK signaling did not substantially affect cell viability both in vitro or in vivo, whereas targeting TEL-JAK2 terminal effectors Bcl-2 and Bcl-XL with ABT-737 was highly effective, synergized with JAK inhibitors and, more importantly, circumvented acquired resistance to JAK Inhibitors (Waibel et al., 8

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2013). Of note, IL-7-dependent aberrant activation of the JAK/STAT signaling pathway, resulting in increased expression of Bcl-2 prosurvival family proteins, is a feature of ETP-ALL (Maude et al., 2015). Bcl-2 expression is also regulated by TYK2 (Tyrosine Kinase 2), a member of the JAK family, and its downstream effector STAT1. TYK2 activation, due to gain of function mutations or autocrine IL-10 receptor signaling, has been reported in both T-ALL cell lines and primary samples (Sanda et al., 2013). Importantly, high expression of Bcl-2 is frequently detected in relapsed/refractory T-ALL, who have an extremely poor outcome, strengthening the importance of targeting Bcl-2 family proteins as a promising therapeutic strategy for these patients (Follini et al., 2019). Several studies reported a higher rate of induction failure of patients with ETP-ALL (Litzow and Ferrando, 2015). In this regard, it has been established that resistance to prednisolone (PSL), a standard component of induction therapy for T-ALL patients, associates to high expression of MEF2C (Myocyte Enhancer Factor 2C), that in turn induces Bcl-2 (Kawashima-Goto et al., 2015). Notably, in ETP-ALL with elevated MEF2C, treatment with ABT-737 restored PLS sensitivity and resulted in a lower number of viable cells compared to single drug treatments (Kawashima-Goto et al., 2015). Beside ETP-ALL, in an in vitro drug screening, Bcl-2 inhibition with ABT-199 has proved to be synergic with the BET (bromodomain and extraterminal) inhibitor JQ1 in primary human relapsed T-ALL cells, and these results were further confirmed in vivo (Peirs et al., 2017). In addition, a comprehensive analysis of deregulated pathways downstream mutant JAK3, that accounts for 16% of T-ALL cases, revealed altered phosphorylation of STAT5 and proteins belonging to the PI3K/AKT/mTOR and MEK/ERK axes, that ultimately culminated in Bcl-2 protein upregulation. Accordingly, inhibiting JAK3, in combination with ABT-199, resulted in synergistic cytotoxicity (Degryse et al., 2018). Additional mechanisms described to induce Bcl-2 overexpression include STAT5B mutations, identified in adult T-ALL patients (Kontro et al., 2014), BRD4-mediated epigenetic regulation in Notch1 mutated and resistant to GSIs T-ALL (Knoechel et al., 2014), and R98S mutation in ribosomal protein L10 (RPL10 R98S) that affects 8% of pediatric T-ALL (Kampen et al., 2019). Of note, in the latter case, ABT-199 displayed effectiveness in vivo in NSG mice injected with R98S pediatric T-ALL samples (Kampen et al., 2019). Although relative to ETP-ALL, more differentiated T-ALL cells are not sensitive to ABT-199, likely due to higher expression levels of Bcl-XL protein, the dual inhibitor ABT-263 demonstrated effectiveness (Dastur et al., 2019). In addition, because sensitization to ABT-263 depends on Mcl-1 levels, that is transcriptionally regulated by mTOR, co-treatment with ABT-263 and the mTOR inhibitor AZD8055 resulted in synergistic activity both in vitro and in vivo (Dastur et al., 2019). These observations are particularly interesting in the context of Notch1 targeting and GSIs resistance, since Notch1 activates PI3K/AKT/mTOR signaling, leading to mTOR activation. Collectively, these data advocate the development of novel therapeutic approaches based on Bcl-2 inhibition for selected TALL subgroups. Because ABT-199 demonstrated substantial clinical response in patients with hematological malignancies (Mihalyova et al., 2018), it received its first global approval in 2016 for the treatment of CLL patients with the 17p deletion. Subsequently, FDA approved ABT-199 in combination with obinutuzumab (anti CD20 antibody) for previously untreated patients with CLL or small lymphocytic lymphoma (SLL), and in combination with 5-azacitidine or decitabine or low-dose cytarabine for the treatment of newlydiagnosed adult AML patients (Deeks, 2016). Currently, several phase I/II clinical trials are investigating ABT-199 in combination with chemotherapeutics for the treatment of adult patients with relapsed/refractory hematologic malignancies, including T-ALL (clinical trials identifier NCT03181126, NCT03504644, NCT03808610, NCT03319901). 7. Metabolic alterations as a therapeutic target in T-ALL The metabolic phenotype of T-ALL cells is the result of both intrinsic genetic mutations and external responses to the microenvironment, as a result of adaptive mechanisms that allow cancer proliferating cells to be supported in their growth and survival, even under stressful conditions (DeBerardinis and Chandel, 2016). The upregulation of glycolysis in aerobic conditions, the so called Warburg effect, has been described as a key pathway to boost the high energy demands and biomass production that cancer cells need to fulfill (Cairns et al., 2011). However, recent studies highlighted that, rather than a switch from glucose oxidation to a higher dependence on glycolysis, cancer cells appear to enhance both glycolysis and oxidative phosphorylation (OXPHOS), as well as other metabolic pathways (Kishton et al., 2016). If on one side, oncogenic signaling in the context of T-ALL has been extensively explored, on the other side, very little is known about metabolic rewiring in these cells and to which extent this is driven by the oncogenic signaling itself. Altered levels of glycolysis were precociously identified in hematological malignancies, as imaging techniques like 18fluorodeoxyglucose positron emission tomography (FDG-PET) were used for detection and diagnosis (Bredella et al., 2005). The overactivation of PI3K/AKT/mTOR pathway stimulates glucose transportation inside the cells through the upregulation of GLUT (glucose transporter) 1 and GLUT4, and it directly promotes glycolysis (Boag et al., 2006), which is also enhanced by other deregulated pathways, such as gain of function of c-Myc and loss of p53. Thus, direct targeting of glycolysis has been tested, through the use of specific inhibitors that target hexokinase (HK), like 2-Deoxy-D-Glucose (2-DG) (Hulleman et al., 2009) and lonidamine (Nista et al., 1985), or pyruvate dehydrogenase kinase (PDK), like dichloroacetate (DCA) (Scatena et al., 2008). However, the potent effects evaluated in vitro still need to be verified in a proper in vivo setting. Recent studies by Kishton et al. showed that oncogenic Notch1 signaling in T-ALL promotes glycolysis but also induces metabolic stress that activates 5′ AMP activated kinase (AMPK). AMPK counteracts mTORC1 activity and boosts oxidative metabolism and mitochondrial complex I activity. Thus, these findings further demonstrated that AMPK-deficiency or pharmacological inhibition of complex I led to T-ALL cell death and reduced disease burden (Kishton et al., 2016). Konopleva and colleagues have shown that targeting OXPHOS through a novel complex 1 inhibitor, IACS-010759 (Molina et al., 2018), proved to have preclinical efficacy in Notch1-driven T-ALL (Baran et al., 2016). Furthermore, combination of IACS-010759 9

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with chemotherapy led to synergistic inhibition of cell proliferation, and proved to be effective in combination with CB-839, a glutaminase inhibitor. This is the result of IACS-010759 activity, that by promoting the glycolytic flux upon OXPHOS inhibition, indirectly induces the use of glutamine to fuel the TCA (tricarboxylic acid cycle) cycle, indicating glutaminolysis as a potential therapeutic target. A key role played by glutamine in Notch1-induced T-ALL was previously described by Herranz and colleagues in the laboratory of A.A. Ferrando (Herranz et al., 2015). In particular, they focused on what was lying beyond GSI treatment resistance in a mouse model of Notch1-induced T-ALL, by performing a wide gene expression profiling analysis. In PTEN-wt cells, glutamine, through glutaminolysis, was described as one of the main carbon sources, particularly by feeding the TCA cycle, while glucose was mostly converted into lactate in these cells. GSI treatment was able to abrogate these conversions and, together with BPTES, a potent glutaminase inhibitor, it showed a synergistic effect. Interestingly, glutaminolysis inhibition, as well as GSI treatment, were abrogated in mice transplanted with Notch1-induced PTEN-null T-ALL cells. This is consistent with the fact that loss of PTEN was able to drive major changes related to increase in anabolic processes, which caused a hyperglycolytic phenotype that compensate glutamine absence to feed metabolic processes. Moreover, in this study, they showed that T-ALL cells rely on autophagy for their survival after Notch1 inhibition, and the antileukemic effect of GSI is stronger when ATG7 is deleted. In addition, another attractive mechanism that can be found in T-ALL and which is connected to a condition of environmental and metabolic stress is the unfolded protein response (UPR) caused by ER stress (Bravo et al., 2013). The ER is involved in a variety of processes including synthesis, folding, modification and transport of proteins, phospholipids and steroids, and it functions also as a Ca++ storage. Any perturbations in at least one of these functions or in the microenvironment can cause a stressful and detrimental effect on the ER itself, known as ER stress, which induces in the cell a series of responses in order to restore the altered homeostasis. UPR signaling is a relevant mechanism that allows cancer cells to maintain their malignancy and gaining therapy resistance, as they require ER integrity to fulfill all the functional requirements they need. It was shown that in T-ALL the downregulation of the UPR pathway leads to extensive apoptosis (DeSalvo et al., 2012). Furthermore, a study showed that CK2 (casein kinase 2) inhibition reduced cell viability through alterations in the UPR pathway (Buontempo et al., 2014). In conclusion, taken together these observations will help elucidate the importance of metabolic aspects in T-ALL to further gain access to its mechanisms and implications, especially concerning patients predicted outcome. An important aspect that needs to be considered is that a key feature of T-ALL metabolism seems to be plasticity, represented by a model in which multiple metabolic pathways are activated and a robust heterogeneity of metabolic phenotypes exists. A major challenge in research will be represented by extensive studies which should include metabolic flux analysis as well as metabolomics experiments, in order to identify specific subtypes targets that will prevent side effects in normal T lymphocytes, a cell population which physiologically activate a metabolic rewiring to support its function. 8. Perspectives T-ALL arises from T-cell precursors at different stages of their maturation and is characterized by molecular genetic subtypes. Much progress has been made in the identification of oncogenic drivers and therapeutic targets in T-ALL, opening several novel opportunities for the development of highly active and less toxic therapies. T-ALL pediatric patients respond quite well to high-dose conventional chemotherapy; however, the clinical response in adults remains yet challenging, and therapeutic options for relapsed T-ALL patients are still very limited. The high survival rates for pediatric T-ALL patients could be the result of a strong treatment of patients. Therefore, given the longterm side effects associated with intensive chemotherapy, risk stratification will be decisive to delineate also pediatric T-ALL treatment protocols, and it will be optimized, based on improved understanding of T-ALL biology. In addition, further reduction of chemotherapy could be also achieved by translation of new molecular and genetic findings into novel targeted therapies for the treatment of T-ALL patients. In this context, several preclinical studies are reporting promising therapeutic effects employing smallmolecule inhibitors targeting specific oncogenic pathways. Novel therapies will require an improved definition of the specific T-ALL patient populations that might benefit from these targeted therapies and that eventually support specific clinical protocols, including combination of both signaling and metabolic inhibitors, along with conventional chemotherapy. Acknowledgments The authors thank Aurelio Valmori for the technical assistance. References Abraham, N., Ma, M.C., Snow, J.W., Miners, M.J., Herndier, B.G., Goldsmith, M.A., 2005. Haploinsufficiency identifies STAT5 as a modifier of IL-7-induced lymphomas. Oncogene 24 (33), 5252–5257. Agnusdei, V., Minuzzo, S., Frasson, C., Grassi, A., Axelrod, F., Satyal, S., Gurney, A., Hoey, T., Seganfreddo, E., Basso, G., Valtorta, S., Moresco, R.M., Amadori, A., Indraccolo, S., 2014. 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