Abstract
BCR-ABL1-negative myeloproliferative neoplasms are classically represented by primary myelofibrosis, polycythemia vera, and essential thrombocythemia. These entities are stem cell-derived clonal disorders characterized by hematopoietic progenitor autonomy or hypersensitivity to cytokines, most of them presenting mutations in Janus kinase 2 (JAK2), calreticulin (CALR), or myeloproliferative leukemia virus oncogene (MPL). Deregulation of pro- and antiapoptotic genes is also claimed as an important mechanism involved in cell resistance to cell death and accumulation of myeloid cells in myeloproliferative neoplasms. Apoptosis, as one of the best-characterized types of programmed cell death, has a clear role in hematopoiesis control. However, the exact pathways affected in BCR-ABL1-negative myeloproliferative neoplasms have not yet been fully clarified. This chapter will explore the modifications affecting programmed cell death pathways involved in myeloid proliferation and how these alterations might be exploited in single or combined targeted therapeutic strategies.
Keywords
- apoptosis
- programmed cell death
- cancer stem cells
- hematological disorders
- cell death mechanisms
- molecular interactions
- cytokine signaling
- cell-cycle inhibitors
1. Introduction
Hematopoiesis is a highly controlled process that ensures the differentiation of the hematopoietic stem cells (HSCs) into lymphoid and myeloid common progenitors and further into all lineages of blood cells [1].
Programmed cell death (PCD) is one of the fundamental mechanisms of an organism’s life cycle that controls every system, including hematopoietic system, based on a precisely tuned signaling network. Apoptosis, the most important type of PCD, maybe because it is the most analyzed type of death to date, is well described in hematopoietic differentiation [2]. Its deregulation in pathological circumstances is potentially deleterious and may influence the fate of the entire organism. Although different other types of PCD were described, apoptosis remains one of the most important processes involved in differentiation and cell survival regulation, while mechanisms as autophagy and necroptosis look like “backup” mechanisms that share some “key players” and diverged from apoptosis at a certain point, to assure the elimination of the malfunctioning system in case of “internal” defect (mutations) or pathogens that inhibit the components of the apoptotic network [3, 4].
Primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocythemia (ET) are classic BCR-ABL1-negative myeloproliferative neoplasms (MPNs) that are stem cell-derived clonal disorders characterized by hematopoietic progenitor autonomy or hypersensitivity to cytokines, driven by acquired somatic mutations in critical pathways, resulting in pathological expansion of the myeloid lineages. In their natural course, MPNs could be exacerbated and transformed into secondary acute myeloid leukemia (sAML) associated with treatment resistance and poor clinical outcome [5].
This chapter will explore the most important modifications affecting programmed cell death pathways involved in myeloid proliferation, and how these alterations might be exploited in single or combined targeted therapeutic strategies in a classic BCR-ABL1-negative MPN.
2. Intrinsic and/or extrinsic apoptotic pathways involved in MPN disease entities
Overall, the hematopoietic homeostasis requires a precise balance between blood cell formation and maintenance of an adequate number of mature cells. Although apoptosis is necessary to prevent the excessive accumulation of cells, the hematopoietic progenitors need to be protected and preserved. A disruption of the homeostatic balance in the hematopoietic system is relevant for many hematological disorders, an increased cell death being involved in the etiology of immune deficiencies and anemia, while an inappropriate resistance to apoptosis might lead to hematological malignancies [6], such as MPNs.
As a particular form of PCD, apoptosis is activated via two convergent pathways: the intrinsic and the extrinsic [7]. The intrinsic signaling pathway is triggered at mitochondrial level in response to various stimuli such as genotoxic agents or growth factor deprivation, and it is mainly regulated by the members of BCL-2 protein family that contain one or more BCL2 homology (BH) domains [8]. These proteins are structurally and functionally classified into three groups. The first group includes the critical effectors of the intrinsic pathway, namely BCL-2 antagonist killer 1 (BAK) and BCL-2-associated X protein (BAX). The second group is represented by the prosurvival BCL-2 proteins (BCL-2, BCL-xL, MCL-1, BCL-W, and A1) that hinder BAK and BAX activation, while the third group comprises several structurally different proteins, known as “BH3-only” proteins (BIM, BID, BAD, BIK, PUMA, and NOXA), which share solely a sequence called BH3-domain [8, 9]. Cellular stress signals are sensed by the “BH3-only” proteins that directly activate BAK and BAX or neutralize the prosurvival proteins. Once activated, through conformational changes, BAK and BAX induce the permeabilization of the mitochondrial membrane with subsequent release of apoptogenic factors, such as cytochrome c and second mitochondrial activator of caspases/direct IAP binding protein with low pI (SMAC/DIABLO). Cytochrome c binds to the apoptotic protease activating factor-1 (APAF-1) and forms the apoptosome, a heptameric complex that activates the initiator caspase-9, followed by activation of the effector caspase-3, caspase-6, and caspase-7 that trigger final events of apoptosis [10, 11].
In the extrinsic apoptotic pathway, caspase activation is elicited at the level of “death receptors” (DR), transmembrane proteins of the tumor necrosis factor (TNF) receptor superfamily typically represented by FAS (CD95), TNF receptors, and TNF-related, apoptosis-inducing ligand (TRAIL) receptors. Through interaction with their corresponding ligands—FASL, TNF-α, and TRAIL, respectively—DR become activated, leading to the recruitment of a death adaptor protein, such as FAS-associated death domain (FADD) or TNFR-1-associated death domain (TRADD). Death adaptors generate a death-inducing signaling complex (DISC), in which procaspase-8 is recruited and activated, the death signal being subsequently transduced to the effector caspases [2, 10].
A very early apoptosis event is the global and rapid mRNA degradation by a mechanism that is not yet completely characterized [12].
Various factors associated with intrinsic and extrinsic apoptotic pathways have been involved in the control of adult hematopoiesis under physiological as well as pathological conditions [2]. In this respect, BCL-2 protein family members play different roles across individual hematopoietic lineages during differentiation and maturation. At the level of HSCs and early myeloid progenitors, MCL-1 is an essential prosurvival factor, being upregulated by stem cell factor and interleukin-3 via JAK/STAT (Janus-activated kinase/signal transducers and activators of transcription) and AKT signaling pathways [13, 14]. During erythropoiesis, erythropoietin (EPO) ensures erythroid progenitor survival, proliferation, and differentiation by acting on its cognate receptor (EPO-R) and inducing JAK2-STAT5 activation that leads to upregulation of BCL-xL [10]. The development, maturation, and survival of megakaryocytes (MKC) is strictly dependent on the presence of both BCL-xL and MCL-1 proteins that are induced by thrombopoietin (TPO) signaling and restrain intrinsic apoptosis, while platelet life span seems to be dictated only by BCL-xL levels [7, 15, 16]. Similarly, MCL-1 is essential for granulocyte progenitor survival and differentiation [16]. On the other hand, the receptor/ligand interactions of the TNF family represent physiological mechanisms that exert a negative regulation in the terminal stages of the hematopoietic differentiation, controlling in this way the size of the expanding hematopoietic clones and maintaining heterogeneity in response to various demands [17].
PV is characterized by erythrocytosis accompanied by a suppressed endogenous EPO production. It often associates thrombocytosis and/or leukocytosis with panmyelosis at bone marrow examination. The pattern of driver mutations is strikingly dominated by
A study that analyzed gene expression profile of granulocytes isolated from PV patients showed an upregulation of protease inhibitors with affinity for proteases inducing apoptosis in neutrophils (e.g., cystatin F and secretory leukocyte protease inhibitor), as well as of several antiapoptotic and survival factors (e.g., p38 MAPK), compared to granulocytes obtained from healthy subjects [19]. Also, unlike the granulocytes of ET patients or normal controls, the granulocytes of PV patients were found to express an increased amount of heat shock protein 70 (HSP70), which counteracts apoptosis at different levels by preventing BAX translocation to mitochondria, inhibiting APAF-1 and procaspase-9 recruitment to apoptosome, and reducing caspase activation. As shown in primary cell cultures, an HSP-70 inhibitor was able to induce apoptosis in the erythroid lineage [20].
Concerning the extrinsic apoptosis pathway, it was found that erythroblasts isolated from PV patients carrying
ET is defined by thrombocytosis associated with normocellular bone marrow and hyperplasia of enlarged MKC. The molecular profile of ET consists of
Before the discovery of
In a study that aimed to characterize the immunophenotypic apoptotic profiles of MKC on bone marrow biopsy samples obtained from MPN patients, it was observed that ET MKC displayed an antiapoptotic pattern, characterized by an overexpression of BCL-xL and a lower expression of BAX, compared to those of PMF patients [24]. Furthermore, Treliński et al. confirmed by flow cytometry the antiapoptotic profile of ET MKC and bone marrow mononuclear cells (BMMC). As opposed to controls, previously untreated ET patients presented significantly lower percentages of apoptotic MKC and BMMCs, when assessed for the number of annexin-V+ and caspase-3+ positive cells. These findings were accompanied by markedly lower BAX levels and BAX/BCL-2 ratios, especially in
Compared to PV and ET, PMF is a more heterogeneous disease, being characterized by clonal myeloproliferation, abnormal cytokine expression, early bone marrow fibrosis, anemia, splenomegaly, extramedullary hematopoiesis, constitutional symptoms, and a lower overall survival rate. On the other hand, during the natural course of the disease PV and ET patients might suffer a conversion into secondary myelofibrosis (MF) that resembles PMF [26, 27]. PMF shares with ET a similar profile of mutations in
Initially, it was suggested that bone marrow MKC in PMF might undergo an increased apoptosis that could be responsible for the release of fibrogenic cytokines [28]. However, further studies have demonstrated that PMF MKC displayed a high proliferative capacity and resistance to apoptosis, explained by the overexpression of BCL-xL [29]. Also, the gene expression analysis of laser-microdissected MKC from PMF patients indicated a tendency toward an overall downregulation of apoptosis-associated genes, especially of
Chronic inflammation sustained by the continuous release of proinflammatory cytokines and chemokines and subsequent bone marrow microenvironment alterations are considered key factors in PMF pathogenesis. The abnormal production of cytokines that occurs both in malignant and nonmalignant cells was related to an increased JAK2-STAT3 activation and was found responsible for the inhibition of apoptosis and increased myeloproliferation, creating an environment that favors MPN clone maintenance and expansion [31, 32]. Recently, it was shown that MF cells downregulated the expression of X-linked inhibitor of apoptosis (XIAP) and mitogen-activated protein kinase 8 (MAPK 8), a necessary component of TNF-mediated apoptosis, via a TNF/TNFR2-dependent autocrine loop. This was considered a mechanism to escape an apoptotic response and to increase NF-κB signaling involved in inflammatory cytokine expression [33].
Overall, these data show the importance of the participation of both intrinsic and extrinsic apoptosis pathways in the pathogenesis of MPNs.
3. Key PCD players in BCR-ABL1-negative MPN entities
Modifications occurred in the regulation of apoptosis, especially in expression of pro- and antiapoptotic genes, have great contribution to the myeloaccumulation in MPNs. Concerning the involvement of other types of PCD in myeloproliferations, few data are available. Some key players are involved in apoptosis regulation and also in autophagy or necroptosis. More often, it is a continuous process from apoptosis, autophagy to necroptosis. Increased death signals and stress levels can switch cell death types in the attempt of eliminating the malfunctioning cells [34, 35].
BCL-2 family of proteins is a very important regulator of apoptosis and, at the same time, is also a negative regulator of BECN1/Beclin-1, a key regulator of autophagy [36, 37]. Autophagy was shown to be a major contributor to chemotherapy resistance in AML [38].
BCL-xL promotes cell survival, such as survival of erythroid cells and platelets, and regulates their lifespan at a steady state. Inhibition of BCL-xL induces profound thrombocytopenia by triggered BAK/BAX-mediated mitochondrial damage, caspase activation, and premature death of MKC [39, 40]. In MPNs, a concerted effect resulted from antiapoptotic BCL-xL over-expression and proapoptotic BNIP-3 downregulation was clearly documented [41].
Bcl-2-associated death promoter (BAD) inhibits antiapoptotic proteins BCL-2 and BCL-xL and is involved in initiating the apoptosis process. In unphosphorylated form, BAD forms heterodimers with BCL-2 and BCL-xL, inhibiting their antiapoptotic functions, and facilitates BAX/BAK activation in response to apoptotic stimuli [42, 43], promoting apoptosis. After activation by phosphorylation, BAD forms a heterodimer with 14-3-3 proteins, releasing BCL-2 that is free to block apoptosis. BAD is a substrate of various kinases, such as AKT, protein kinase A (PKA), and c-Jun NH2-terminal kinase (JNK).
Gene expression studies on CD34+ cells and peripheral leukocytes isolated from ET and PMF patients indicated that mRNA levels of
Studies focused on the apoptosis deregulation in PV identified an increased expression of
Survivin is one of the inhibitors of apoptosis proteins (IAPs) that regulate cell death through mitochondrial route by restricting the IAP-inhibitor DIABLO protein and preventing it from activating caspase-9. A greater proportion of myeloproliferative MKC express survivin compared to its reciprocal inhibitor, DIABLO. Survivin seems to be the key mediator of the MKC survival signature in the MPNs and might be a potential therapeutic target [41]. Recently, new evidence suggested that survivin may be involved in the evasion of cell death by manipulation of autophagy [49].
BNIP-3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3), a proapoptotic mitochondrial protein belonging to the BCL-2 family, is activated under hypoxic conditions with hypoxia-inducible factor (HIF-1α) in normal and cancer tissues. BNIP-3 is involved in the induction of hypoxic necrosis in tumors because it activates caspase-independent necrosis-like cell death by opening the mitochondrial permeability transition pore. In MPNs, BNIP-3 expression is reportedly low and this might indicate that the increased bone marrow cellularity is not only because of proliferative signaling but also due to decreased apoptosis [50, 51].
CALR is a multifunctional endoplasmic reticulum (ER) chaperone involved in the quality control of N-glycosylated proteins, calcium storage, and immune responses [27, 52]. In relation to apoptosis, CALR is implicated in the specific activation of caspase-8, BAX, and BAK, and also in the BCL-2 cleavage [53].
Caspase-8, a key factor in the extrinsic pathway, together with caspase-9, a key factor in the intrinsic pathway, is implicated in regulating MKC turnover [41].
Immunohistochemistry studies showed that the percentage of MKC positive for caspase-8 is higher in MPNs in comparison with controls, suggesting that MKC in MPN tend to counteract the survival advantages acquired through inhibition of the intrinsic apoptotic pathway by activating the caspase-8-mediated extrinsic apoptotic cascade[41].
Caspase-9 is an inducible proapoptotic molecule, which acts relatively late in apoptosis signaling becoming less susceptible to inhibition by apoptosis inhibitors [56]. Caspase-9 is an apoptotic initiator caspase in MKC and platelets being necessary for their efficient death, and it is not required for platelet generation and function, as it was previously thought. Thus, caspase-9 loss is associated with an increased MKC proliferative capacity. In MPNs, especially in the
SMAC/DIABLO controls apoptosis by negatively regulating IAPs and by activating caspases. Recently, it was shown that silencing of SMAC/DIABLO caused decreased levels of phospholipids, suggesting that besides proapoptotic functions, SMAC/DIABLO have nonapoptotic lipid synthesis-related function essential for cancer growth and development. Therefore, it was assumed that SMAC/DIABLO could be a promising therapeutic target in cancer [57]. On the other hand, SMAC/DIABLO downregulation was found to be associated with progressive disease and poor survival rate in hematologic malignancies, and DIABLO/SMAC mimetics were proposed as a potential adjunct therapy to enhance DIABLO levels in MPN MKC [41]. More studies are necessary to establish the proper therapeutic options in the light of the new role of SMAC/DIABLO in the phospholipid synthesis.
The tumor suppressor gene
Cell surface death receptor-ligand interaction, such as FASL binding FAS, TRAIL binding death receptor 5 (DR5) or TNFα binding TNFR1, executes extrinsic pathway apoptosis.
The two major necroptotic death effector complexes, the necrosome and ripoptosome, are induced by TNFR1 and toll like receptor 3 (TLR3) signaling, respectively [60]. IFN-R activation, primarily by type-I IFN, is believed to involve a caspase- and FADD-independent, receptor-interacting protein kinase (RIP) 3-dependent mode of cell death via the formation of the necrosome. Following IFN-R activation, JAK/STAT signaling and the activity of RNA-responsive protein kinase (PKR), upstream of RIP1/RIP3 necrosome formation is essential. TNFα binding to TNFR1 causes recruitment of TRADD and RIP1 via their death domains resulting in the prosurvival complex I, which is stabilized by TNFα-bound TNFR2-TRAF2. Internalization of the TNFR1-TRADD-RIP1 complex is required for recruitment of caspase-8 and FADD, necessary for apoptosis. This is therefore a major cell death checkpoint as the absence of NF-κB activation and prosurvival signaling results in proapoptotic complexes or, alternatively, the pronecroptotic complex known as the necrosome. The ripoptosome consists of FADD, cFLIP and caspase-8 and allows necroptosis to prevail if active cleavage of RIP1 by caspase-8 is prevented by cFLIPL. In MPN, it was shown that blocking TNFR2 but not TNFR1 selectively inhibits MPN cells over normal ones and the process involves XIAP, cIAP, and MAPK8 as key mediators of these differential responses to TNF. These data support the potential therapeutic use of cIAP inhibitors and selective TNFR2 inhibitors in the treatment of MF [33].
The TLR3-induced pathway converges with the TNFR1-induced pathway at the necrosome. The execution phase of necroptosis starts with interaction between RIP1 and RIP3. Following stabilization of the RIP1-RIP3 complex, mixed-lineage kinase domain-like protein (MLKL) is recruited to form a functional necrosome. MLKL activated upon phosphorylation by RIP3 results in the translocation of the MLKL necrosome to the plasma membrane, necroptotic membrane disruption, and release of liposomes containing phosphatidylinositol phosphates (PIPs). This permeabilization, combined with MLKL-mediated calcium or sodium influx ion-pore dysregulation, characterizes the model proposed for necroptosis execution [60]. Human cancers, including MPN and their exacerbated form, sAML, are known for eluding apoptosis; therefore, therapeutic induction of necroptosis may represent a better strategy for an efficient treatment. A series of compounds have been shown to trigger necroptosis, particularly inhibitors of RIP1, RIP3 or MLKL, in leukemia cells; however, a deeper understanding of the signaling network that regulates this type of PCD is still necessary [35].
4. Major signaling pathways involved in apoptotic failure in molecular subgroups of MPN
The constitutive activation of JAK-STAT pathway is a common feature of MPNs irrespective of driving mutation, being observed even in so-called “triple-negative MPNs” that lack known
In addition, loss-of-function or neomorph mutations in genes that are involved in epigenetic regulation, splicing, and signaling can act as disease modifiers by cooperating with MPN driver mutations [52].
The JAK/STAT is the major pathway (Figure 1a) involved in MPN pathology [64, 65, 66].
The MAPK/ERK signaling pathway activation (Figure 1b) is required in MKC differentiation, with TPO as signal for induced maturation via MPL receptor [71].
An increased activation of RAS/RAF/ERK pathway was showed in MPN patients, especially in erythroid precursor cells and MKC (Figure 1b). It was shown that ERK is constitutively activated by the
The PI3K/AKT signaling pathway (Figure 1c) may be activated by the
The activation of AKT upregulates BCL-xL and inactivates BAD, suppressing apoptosis and promoting cell survival. This was observed in MPN patients, where the activation of pAKT was higher in MKC and associated with the inhibition of MKC apoptosis [50]. pAKT is also known to induce activation of BNIP-3 and caspase-9 through mammalian target of rapamycin (mTOR)—a serine/threonine kinase that is an effector protein of AKT—via activation of HIF-1. Data related to BNIP-3 expression are conflicting; some groups reported a reduced BNIP-3 expression [30], whereas others have shown its upregulation in MPNs [50]. In the study of Koopmans et al., the immunohistochemical expression of BNIP-3, with proapoptotic function, was lower in total bone marrow cells of ET, PV, and PMF patients, compared with the control group. This suggests that a decreased apoptosis might also contribute to the increased bone marrow cellularity observed in MPNs. However, in contrast to total bone marrow cells, the MKC of MPN patients were found to display a high level of BNIP-3 [50]. On the other hand, the most pronounced reductions in BNIP-3 were observed in PMF, suggesting a loss of proapoptotic potential during progression to the “accelerated” phase of MPNs [80].
Noncanonical
Taking into account the above-mentioned cellular and molecular effects of MPN driver mutations, we can assume that megakaryocytic and erythroid progenitor expansion in MPNs results from a combination of increased proliferation and attenuated apoptosis.
5. PCD resistance in myeloid proliferation exploited in single or combined targeted therapeutic strategies
As shown previously, deregulation of the JAK/STAT pathway is central to MPN development and is driven in most cases by activating mutations in
Understanding molecular mechanisms of MPN pathogenesis has stimulated drug development in the field.
Reduction of thrombotic risk is the major goal of therapy in patients with PV and ET, and hydroxyurea (HU) is normally the first-line drug for achieving cytoreduction [98, 99]. In addition, most patients should receive aspirin, if they have no contraindications. In PV, maintaining hematocrit values <45% is an important therapeutic target. Second-line drugs of choice are interferon-α (IFNα) and busulfan [99].
The clinical efficacy of IFNα has been reported since 30 years ago and was improved with the development of pegylated forms [100, 101]. Furthermore, significant reductions of the
RUX showed efficiency in spleen size reduction and symptomatology alleviation, improving quality of life, and overall survival; however, no significant decrease in allele burden was achieved [11, 111, 112]. RUX effects on the malignant clone are modest, side effects (such as anemia and thrombocytopenia) are reported, and drug resistance may appear. Other therapeutic strategies have been developed; they include the discovery of new inhibitors that target specifically mutant JAK2 and the combination of current therapies with other molecules that inhibit components of signaling pathway [105].
Early studies provided some evidence for the increased resistance to apoptosis of PV erythroid progenitor cells: overexpression of BCL-xL in the absence of EPO and a higher sensitivity to the antiapoptotic growth factor IGF-1 [113]. Moreover, Zeuner et al. have shown that erythroid precursors in PV patients with average and high
In susceptible cells, apoptosis is caused by exposure to a JAK inhibitor, which leads to dephosphorylation of BAD, enabling BAD to bind and sequester the antiapoptotic protein BCL-xL. On the other side, in potent cells, RAS effector pathways keep BAD phosphorylation in the presence of JAK inhibitors, maintaining a specific dependence on BCL-xL for survival. So, downstream regulation of BCL-xL, more precisely BCL-xL inhibition, might be the key against resistance to JAK inhibition by either co-inhibition of JAK and RAS effector in AKT and ERK pathways or by direct inhibition of BCL-xL inducing apoptosis [116].
At present, there are over 1500 clinical trials evaluating various drug effects on myeloproliferative neoplasms registered at clinicaltrials.gov. Some of them might be successful due to targeting different apoptotic pathways or by targeting simultaneously different types of PCD.
Plitidepsin is a synthetically produced anticancer agent [117], a cyclodepsipeptide related to didemnins, commercialized as Aplidin® (PharmaMar, S.A., Madrid, Spain). Plitidepsin induces dose-dependent cell-cycle arrest and an acute apoptotic process. These effects rely on the induction of early oxidative stress, the rapid activation of Rac1 GTPase, and the sustained activation of JNK and p38/MAPK, which finally result in caspase-dependent apoptosis [118, 119]. JNK phosphorylation can be seen as early as 5–10 minutes after exposure to the compound. The activation of JNK and p38/MAPK is associated with increase in reactive oxygen species and a decrease in reduced form of glutathione [120].
Recent studies have led researchers to hypothesize that the primary target of plitidepsin could be the eukaryotic elongation factor 1A2 (eEF1A2), which is overexpressed in tumors and supports tumor cell proliferation while inhibiting apoptosis [121]. eEF1A2 seems to be an interesting target for therapy and may also be a biomarker predicting drug sensitivity. Aplidin®/Plitidepsin was investigated for its effect (safety and tolerability) on bone marrow or peripheral blood cells as well as assessed the response rate in patients with PMF, post-PV MF, or post-ET MF, in phase II/open label single agent clinical trial (NCT01149681). Although the drug was well tolerated, the trial was prematurely terminated due to the low response rate [122].
Navitoclax is an orally active, synthetic small molecule and an antagonist of the apoptosis suppressor proteins BCL-2, BCL-xL, and BCL-w, which are frequently overexpressed in a wide variety of cancers, including myeloid ones, and are linked to drug resistance. Inhibition of these apoptosis suppressors prevents their binding to the apoptotic effectors BAX and BAK proteins, thereby triggering apoptotic processes in cells overexpressing BCL-2, BCL-xL, and BCL-w. This eventually reduces tumor cell proliferation. Navitoclax (ABT-263) and RUX are currently evaluated in combination for efficacy, safety, and tolerability on spleen volume as assessed by magnetic resonance imaging (MRI) in participants with MF in a phase II/open label clinical trial (NCT03222609).
Obatoclax (GX15–070) is a BH3-mimetic designed to target and counteract antiapoptotic BCL-2 proteins. Obatoclax is an MCL-1 antagonist [123] and downregulates p53, and it has a dual mechanism of action, being capable to induce apoptosis or autophagy [124]. On the other side, obatoclax accumulates in lysosomes inducing their alkalinization and inhibiting their function [125]. Parikh et al. conducted a multicenter, open-label, noncomparative phase II study (NCT00360035) of obatoclax in patients with MF. Unfortunately, obatoclax exhibited no significant clinical activity at the tested dose and schedule [126].
Other phase I trial (NCT02436135) investigated the combination of RUX with idelalisib, a PI3K delta inhibitor, as therapy for intermediate to high-risk PMF, post-PV MF, or post-ET MF with progressive or relapsed disease [127].
PIM inhibitors have shown preclinical synergy with JAK inhibitors, as well as the ability to overcome JAK inhibitor resistance in MPN cell lines. PIM regulate JAK/STAT signaling and are involved in oncogenesis through phosphorylation of cell cycle regulators, activation of antiapoptotic proteins, and enhancement of MYC expression [97]. A phase 1b study of RUX plus PIM inhibitor PIM447, or RUX plus CDK4/6 inhibitor ribociclib (LEE011), or the combination of all three is underway in several non-U.S. countries (NCT02370706).
As PI3K/AKT/mTOR signaling is markedly activated in MPNs, small molecule inhibitors of the proteins involved in this pathway have been tested in MF with promising results. Thus, mTOR inhibitor everolimus, as single therapeutic agent, was able to induce responses, in terms of reducing constitutional symptoms and the degree of leukocytosis, thrombocytosis, and anemia, in 23% of patients in a phase I/II clinical trial. Due to the preclinically proved synergic effects of PI3K/AKT/mTOR inhibitors and JAK inhibitors, several clinical studies were initiated: PI3K inhibitor TGR-1202 in combination with RUX (NCT02493530), PI3K inhibitor buparlisib with RUX (NCT01730248), PI3K inhibitor INCB050465 and RUX (NCT02718300), and selective PI3Kδ inhibitor TGR-1202 and RUX (NCT02493530). Preliminary results of buparlisib and RUX phase 1b study indicated that this drug association was well tolerated, and ≥ 50% reduction in splenomegaly was observed in 70% of JAK-inhibitor naive patients and 54% of patients who did not previously respond to JAK2 inhibitor monotherapy [97, 127].
RAF/MEK/ERK pathway is another signaling cascade activated in MPNs by the increased JAK/STAT signaling. Therefore, MEK inhibitors were tested in different murine models, either alone or in combination with JAK inhibitors, showing a decrease in bone marrow fibrosis, inhibition of malignant cell growth, and HSC function recovery, associated with a prolonged survival. Moreover, a new trial that combines the MEK inhibitor selumetinib with the DNA hypomethylating agent azacitidine is soon expected [97].
Preclinical studies reveal a central role for tumor necrosis factor alfa (TNF-α) in promoting clonal dominance of
SMAC-mimetics are novel apoptosis-inducing agents that stimulate the ubiquitinylation and proteasomal degradation of cellular inhibitors of apoptosis (IAPs) [102], proteins that play an important role in tumor cell resistance to cytotoxicity mediated by TNF superfamily cytokines. These agents have been shown to sensitize cancer cells to TNF family-induced apoptosis [103]. Results from a phase II trial of the SMAC-mimetic LCL-161 in patients with intermediate or high-risk MF intolerant of, ineligible for, or relapsed/refractory to JAK inhibitors were recently presented. Six of sixteen evaluated patients (38%) had objective responses, obtaining clinical improvement and in one case cytogenetic remission [128].
Resistance of hematologic malignancies to PCD significantly limits the efficacy of chemotherapy. As the majority of chemotherapeutic drugs trigger apoptosis, the observed resistance may indicate that novel therapeutic strategies to reactivate nonapoptotic PCD or at least combined therapeutic strategies able to attack simultaneously different mechanisms might be better approaches to eradicate malignant cells.
6. Conclusion
Deregulation of pro- and anti-PCD genes involved in cell resistance to cell death and accumulation of myeloid cells in MPNs is continuously clarified by intense exploration of the modifications affecting different types of PCD pathways involved in myeloid proliferation. At the same time, comprehension of the network of signaling pathways involved in etiology and drug resistance of these disorders facilitate a more efficient exploitation of the knowledge, using combined and synergic, targeted therapeutic strategies.
Acknowledgments
We gratefully acknowledge the funding from the project Competitiveness Operational Programme (COP) A1.1.4. ID: P_37_798 MyeloAL-EDiaProT, Contract 149/26.10.2016, (SMIS: 106774), MyeloAL Project.
References
- 1.
Orkin SH, Zon LI. Hematopoiesis: An evolving paradigm for stem cell biology. Cell. 2008; 132 (4):631-644. DOI: 10.1016/j.cell.2008.01.025 - 2.
Sarvothaman S, Undi RB, Pasupuleti SR, Gutti U, Gutti RK. Apoptosis: Role in myeloid cell development. Blood Research. 2015; 50 (2):73-79. DOI: 10.5045/br.2015.50.2.73 - 3.
Ishida S, Akiyama H, Umezawa Y, Okada K, Nogami A, Oshikawa G, et al. Mechanisms for mTORC1 activation and synergistic induction of apoptosis by ruxolitinib and BH3 mimetics or autophagy inhibitors in JAK2-V617F-expressing leukemic cells including newly established PVTL-2. Oncotarget. 2018; 9 (42):26834-26851. DOI: 10.18632/oncotarget.25515 - 4.
Murphy JM, Silke J. Ars Moriendi; the art of dying well—New insights into the molecular pathways of necroptotic cell death. EMBO Reports. 2014; 15 (2):155-164. DOI: 10.1002/embr.201337970 - 5.
Spivak JL. Myeloproliferative neoplasms. The New England Journal of Medicine. 2017; 376 (22):2168-2181. DOI: 10.1056/NEJMra1406186 - 6.
Opferman JT, Kothari A. Anti-apoptotic BCL-2 family members in development. Cell Death and Differentiation. 2018; 25 (1):37-45. DOI: 10.1038/cdd.2017.170 - 7.
McArthur K, Chappaz S, Kile BT. Apoptosis in megakaryocytes and platelets: The life and death of a lineage. Blood. 2018; 131 (6):605-610. DOI: 10.1182/blood-2017-11-742684 - 8.
Gores GJ, Kaufmann SH. Selectively targeting Mcl-1 for the treatment of acute myelogenous leukemia and solid tumors. Genes & Development. 2012; 26 (4):305-311. DOI: 10.1101/gad.186189.111 - 9.
Kile BT. The role of apoptosis in megakaryocytes and platelets. British Journal of Haematology. 2014; 165 (2):217-226. DOI: 10.1111/bjh.12757 - 10.
Testa U. Apoptotic mechanisms in the control of erythropoiesis. Leukemia. 2004; 18 (7):1176-1199. DOI: 10.1038/sj.leu.2403383 - 11.
Tognon R, Nunes Nde S, Castro FA. Apoptosis deregulation in myeloproliferative neoplasms. Einstein (Sao Paulo). 2013; 11 (4):540-544 - 12.
Thomas MP, Liu X, Whangbo J, McCrossan G, Sanborn KB, Basar E, et al. Apoptosis triggers specific, rapid, and global mRNA decay with 3' uridylated intermediates degraded by DIS3L2. Cell Reports. 2015; 11 (7):1079-1089. DOI: 10.1016/j.celrep.2015.04.026 - 13.
Craig RW. MCL1 provides a window on the role of the BCL2 family in cell proliferation, differentiation and tumorigenesis. Leukemia. 2002; 16 (4):444-454. DOI: 10.1038/sj.leu.2402416 - 14.
Opferman JT. Life and death during hematopoietic differentiation. Current Opinion in Immunology. 2007; 19 (5):497-502. DOI: 10.1016/j.coi.2007.06.002 - 15.
Kodama T, Hikita H, Kawaguchi T, Shigekawa M, Shimizu S, Hayashi Y, et al. Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages. Cell Death and Differentiation. 2012; 19 (11):1856-1869. DOI: 10.1038/cdd.2012.88 - 16.
Kollek M, Muller A, Egle A, Erlacher M. Bcl-2 proteins in development, health, and disease of the hematopoietic system. The FEBS Journal. 2016; 283 (15):2779-2810. DOI: 10.1111/febs.13683 - 17.
Mizrahi K, Askenasy N. Physiological functions of TNF family receptor/ligand interactions in hematopoiesis and transplantation. Blood. 2014; 124 (2):176-183. DOI: 10.1182/blood-2014-03-559641 - 18.
Rumi E, Cazzola M. Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms. Blood. 2017; 129 (6):680-692. DOI: 10.1182/blood-2016-10-695957 - 19.
Pellagatti A, Vetrie D, Langford CF, Gama S, Eagleton H, Wainscoat JS, et al. Gene expression profiling in polycythemia vera using cDNA microarray technology. Cancer Research. 2003; 63 (14):3940-3944 - 20.
Gallardo M, Barrio S, Fernandez M, Paradela A, Arenas A, Toldos O, et al. Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera. Molecular Cancer. 2013; 12 :142. DOI: 10.1186/1476-4598-12-142 - 21.
Zeuner A, Pedini F, Signore M, Ruscio G, Messina C, Tafuri A, et al. Increased death receptor resistance and FLIPshort expression in polycythemia vera erythroid precursor cells. Blood. 2006; 107 (9):3495-3502. DOI: 10.1182/blood-2005-07-3037 - 22.
Tognon R, Gasparotto EP, Leroy JM, Oliveira GL, Neves RP, Carrara Rde C, et al. Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases. Journal of Clinical Pathology. 2011; 64 (1):75-82. DOI: 10.1136/jcp.2010.080895 - 23.
Tenedini E, Fagioli ME, Vianelli N, Tazzari PL, Ricci F, Tagliafico E, et al. Gene expression profiling of normal and malignant CD34-derived megakaryocytic cells. Blood. 2004; 104 (10):3126-3135. DOI: 10.1182/blood-2003-07-2597 - 24.
Florena AM, Tripodo C, Di Bernardo A, Iannitto E, Guarnotta C, Porcasi R, et al. Different immunophenotypical apoptotic profiles characterise megakaryocytes of essential thrombocythaemia and primary myelofibrosis. Journal of Clinical Pathology. 2009; 62 (4):331-338. DOI: 10.1136/jcp.2007.054353 - 25.
Trelinski J, Chojnowski K, Cebula-Obrzut B, Smolewski P. Impaired apoptosis of megakaryocytes and bone marrow mononuclear cells in essential thrombocythemia: Correlation with JAK2V617F mutational status and cytoreductive therapy. Medical Oncology. 2012; 29 (4):2388-2395. DOI: 10.1007/s12032-012-0202-3 - 26.
Tefferi A. Primary myelofibrosis: 2014 update on diagnosis, risk-stratification, and management. American Journal of Hematology. 2014; 89 (9):915-925. DOI: 10.1002/ajh.23703 - 27.
Cahu X, Constantinescu SN. Oncogenic drivers in myeloproliferative neoplasms: From JAK2 to calreticulin mutations. Current Hematologic Malignancy Reports. 2015; 10 (4):335-343. DOI: 10.1007/s11899-015-0278-x - 28.
Thiele J, Lorenzen J, Manich B, Kvasnicka HM, Zirbes TK, Fischer R. Apoptosis (programmed cell death) in idiopathic (primary) osteo−/myelofibrosis: Naked nuclei in megakaryopoiesis reveal features of para-apoptosis. Acta Haematologica. 1997; 97 (3):137-143. DOI: 10.1159/000203671 - 29.
Ciurea SO, Merchant D, Mahmud N, Ishii T, Zhao Y, Hu W, et al. Pivotal contributions of megakaryocytes to the biology of idiopathic myelofibrosis. Blood. 2007; 110 (3):986-993. DOI: 10.1182/blood-2006-12-064626 - 30.
Theophile K, Hussein K, Kreipe H, Bock O. Expression profiling of apoptosis-related genes in megakaryocytes: BNIP3 is downregulated in primary myelofibrosis. Experimental Hematology. 2008; 36 (12):1728-1738. DOI: 10.1016/j.exphem.2008.07.011 - 31.
Kleppe M, Kwak M, Koppikar P, Riester M, Keller M, Bastian L, et al. JAK–STAT Pathway Activation in Malignant and Nonmalignant Cells Contributes to MPN Pathogenesis and Therapeutic Response. 2015; 5 (3):316-331. DOI:10.1158/2159-8290.CD-14-0736%J Cancer Discovery - 32.
Desterke C, Martinaud C, Ruzehaji N, Le Bousse-Kerdiles MC. Inflammation as a keystone of bone marrow stroma alterations in primary myelofibrosis. Mediators of Inflammation. 2015; 2015 :415024. DOI: 10.1155/2015/415024 - 33.
Heaton WL, Senina AV, Pomicter AD, Salama ME, Clair PM, Yan D, et al. Autocrine Tnf signaling favors malignant cells in myelofibrosis in a Tnfr2-dependent fashion. Leukemia. 2018; 32 :2399-2411. DOI: 10.1038/s41375-018-0131-z - 34.
Long JS, Ryan KM. New frontiers in promoting tumour cell death: Targeting apoptosis, necroptosis and autophagy. Oncogene. 2012; 31 (49):5045-5060. DOI: 10.1038/onc.2012.7 - 35.
Huang X, Xiao F, Li Y, Qian W, Ding W, Ye X. Bypassing drug resistance by triggering necroptosis: Recent advances in mechanisms and its therapeutic exploitation in leukemia. Journal of Experimental & Clinical Cancer Research. 2018; 37 (1):310. DOI: 10.1186/s13046-018-0976-z - 36.
Hardwick JM, Soane L. Multiple functions of BCL-2 family proteins. Cold Spring Harbor Perspectives in Biology. 2013; 5 (2):1-22. DOI: 10.1101/cshperspect.a008722 - 37.
Visconte V, Przychodzen B, Han Y, Nawrocki ST, Thota S, Kelly KR, et al. Complete mutational spectrum of the autophagy interactome: A novel class of tumor suppressor genes in myeloid neoplasms. Leukemia. 2017; 31 (2):505-510. DOI: 10.1038/leu.2016.295 - 38.
Sehgal AR, Konig H, Johnson DE, Tang D, Amaravadi RK, Boyiadzis M, et al. You eat what you are: autophagy inhibition as a therapeutic strategy in leukemia. Leukemia. 2015; 29 (3):517-525. DOI: 10.1038/leu.2014.349 - 39.
Adams JM, Cory S. The BCL-2 arbiters of apoptosis and their growing role as cancer targets. Cell Death and Differentiation. 2018; 25 (1):27-36. DOI: 10.1038/cdd.2017.161 - 40.
White MJ, Schoenwaelder SM, Josefsson EC, Jarman KE, Henley KJ, James C, et al. Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function. Blood. 2012; 119 (18):4283-4290. DOI: 10.1182/blood-2011-11-394858 - 41.
Malherbe JA, Fuller KA, Mirzai B, Kavanagh S, So CC, Ip HW, et al. Dysregulation of the intrinsic apoptotic pathway mediates megakaryocytic hyperplasia in myeloproliferative neoplasms. Journal of Clinical Pathology. 2016; 69 (11):1017-1024. DOI: 10.1136/jclinpath-2016-203625 - 42.
Zhang J, Huang K, O'Neill KL, Pang X, Luo X. Bax/Bak activation in the absence of Bid, Bim, Puma, and p53. Cell Death & Disease. 2016; 7 :e2266. DOI: 10.1038/cddis.2016.167 - 43.
Fletcher JI, Meusburger S, Hawkins CJ, Riglar DT, Lee EF, Fairlie WD, et al. Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax. Proceedings of the National Academy of Sciences of the United States of America. 2008; 105 (47):18081-18087. DOI: 10.1073/pnas.0808691105 - 44.
Tognon R, Gasparotto EP, Neves RP, Nunes NS, Ferreira AF, Palma PV, et al. Deregulation of apoptosis-related genes is associated with PRV1 overexpression and JAK2 V617F allele burden in essential thrombocythemia and myelofibrosis. Journal of Hematology & Oncology. 2012; 5 :2. DOI: 10.1186/1756-8722-5-2 - 45.
Gasparotto EPL, Tognon R, Ferreira AF, Oliveira GLV, Palma PVB, Zanichelli MA, et al. Deregulated expression of A1, Bcl-2, Bcl-xL, and Mcl-1 antiapoptotic proteins and Bid, Bad, and Bax proapoptotic genes in polycythemia vera patients. Brazilian Journal of Pharmaceutical Sciences. 2011; 47 :873-886 - 46.
Rubert J, Qian Z, Andraos R, Guthy DA, Radimerski T. Bim and Mcl-1 exert key roles in regulating JAK2V617F cell survival. BMC Cancer. 2011; 11 :24. DOI: 10.1186/1471-2407-11-24 - 47.
Zhao R, Follows GA, Beer PA, Scott LM, Huntly BJ, Green AR, et al. Inhibition of the Bcl-xL deamidation pathway in myeloproliferative disorders. The New England Journal of Medicine. 2008; 359 (26):2778-2789. DOI: 10.1056/NEJMoa0804953 - 48.
Malherbe JA, Fuller KA, Arshad A, Nangalia J, Romeo G, Hall SL, et al. Megakaryocytic hyperplasia in myeloproliferative neoplasms is driven by disordered proliferative, apoptotic and epigenetic mechanisms. Journal of Clinical Pathology. 2016; 69 (2):155-163. DOI: 10.1136/jclinpath-2015-203177 - 49.
Humphry NJ, Wheatley SP. Survivin inhibits excessive autophagy in cancer cells but does so independently of its interaction with LC3. Biology Open. 2018; 7 (10):1-7. DOI: 10.1242/bio.037374 - 50.
Koopmans SM, Schouten HC, van Marion AM. Anti-apoptotic pathways in bone marrow and megakaryocytes in myeloproliferative neoplasia. Pathobiology. 2014; 81 (2):60-68. DOI: 10.1159/000356187 - 51.
Giatromanolaki A, Koukourakis MI, Sowter HM, Sivridis E, Gibson S, Gatter KC, et al. BNIP3 expression is linked with hypoxia-regulated protein expression and with poor prognosis in non-small cell lung cancer. Clinical Cancer Research. 2004; 10 (16):5566-5571. DOI: 10.1158/1078-0432.CCR-04-0076 - 52.
Vainchenker W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017; 129 (6):667-679. DOI: 10.1182/blood-2016-10-695940 - 53.
Wang WA, Groenendyk J, Michalak M. Calreticulin signaling in health and disease. The International Journal of Biochemistry & Cell Biology. 2012; 44 (6):842-846. DOI: 10.1016/j.biocel.2012.02.009 - 54.
Mondet J, Park JH, Menard A, Marzac C, Carillo S, Pourcelot E, et al. Endogenous megakaryocytic colonies underline association between megakaryocytes and calreticulin mutations in essential thrombocythemia. Haematologica. 2015; 100 (5):e176-e178. DOI: 10.3324/haematol.2014.118927 - 55.
Kominami K, Nakabayashi J, Nagai T, Tsujimura Y, Chiba K, Kimura H, et al. The molecular mechanism of apoptosis upon caspase-8 activation: quantitative experimental validation of a mathematical model. Biochimica et Biophysica Acta. 2012; 1823 (10):1825-1840. DOI: 10.1016/j.bbamcr.2012.07.003 - 56.
Straathof KC, Pule MA, Yotnda P, Dotti G, Vanin EF, Brenner MK, et al. An inducible caspase 9 safety switch for T-cell therapy. Blood. 2005; 105 (11). DOI: 4247-4254 DOI:10.1182/blood-2004-11-4564 - 57.
Paul A, Krelin Y, Arif T, Jeger R, Shoshan-Barmatz V. A new role for the mitochondrial pro-apoptotic protein SMAC/Diablo in phospholipid synthesis associated with tumorigenesis. Molecular Therapy. 2018; 26 (3):680-694. DOI: 10.1016/j.ymthe.2017.12.020 - 58.
Song H, Hollstein M, Xu Y. p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM. Nature Cell Biology. 2007; 9 (5):573-580. DOI: 10.1038/ncb1571 - 59.
Chen S, Gao R, Yao C, Kobayashi M, Liu SZ, Yoder MC, et al. Genotoxic stresses promote clonal expansion of hematopoietic stem cells expressing mutant p53. Leukemia. 2018; 32 (3):850-854. DOI: 10.1038/leu.2017.325 - 60.
Hanson B. Necroptosis: A new way of dying? Cancer Biology & Therapy. 2016; 17 (9):899-910. DOI: 10.1080/15384047.2016.1210732 - 61.
Diaconu C, Mambet C, Necula LG, Gurban P, Matei L, Aldea-Pitica IM, et al. Triple negative myeloproliferative neoplasms-sometimes driver mutations stay low-key in plain sight. Romanian Biotechnological Letters. 2018; 23 (4):13750-13760. DOI: 10.26327/RBL2017.12 - 62.
Mambet C, Matei L, Necula LG, Diaconu CC. A link between the driver mutations and dysregulated apoptosis in BCR-ABL1 negative myeloproliferative neoplasms. Journal of Immunoassay & Immunochemistry. 2016; 37 (4):331-345. DOI: 10.1080/15321819.2016.1152276 - 63.
Vainchenker W, Leroy E, Gilles L, Marty C, Plo I, Constantinescu SN. JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders. F1000Res. 2018; 7 :82. DOI: 10.12688/f1000research.13167.1 - 64.
James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005; 434 (7037):1144-1148. DOI: 10.1038/nature03546 - 65.
Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. The New England Journal of Medicine. 2005; 352 (17):1779-1790. DOI: 10.1056/NEJMoa051113 - 66.
Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005; 7 (4):387-397. DOI: 10.1016/j.ccr.2005.03.023 - 67.
Mustjoki S, Borze I, Lasho TL, Alitalo R, Pardanani A, Knuutila S, et al. JAK2V617F mutation and spontaneous megakaryocytic or erythroid colony formation in patients with essential thrombocythaemia (ET) or polycythaemia vera (PV). Leukemia Research. 2009; 33 (1):54-59. DOI: 10.1016/j.leukres.2008.07.008 - 68.
Dusa A, Staerk J, Elliott J, Pecquet C, Poirel HA, Johnston JA, et al. Substitution of pseudokinase domain residue Val-617 by large non-polar amino acids causes activation of JAK2. The Journal of Biological Chemistry. 2008; 283 (19):12941-12948. DOI: 10.1074/jbc.M709302200 - 69.
Yasuda M, Han JW, Dionne CA, Boyd JM, Chinnadurai G. BNIP3alpha: a human homolog of mitochondrial proapoptotic protein BNIP3. Cancer Research. 1999; 59 (3):533-537 - 70.
Azad MB, Chen Y, Henson ES, Cizeau J, McMillan-Ward E, Israels SJ, et al. Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3. Autophagy. 2008; 4 (2):195-204 - 71.
Rouyez MC, Boucheron C, Gisselbrecht S, Dusanter-Fourt I, Porteu F. Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway. Molecular and Cellular Biology. 1997; 17 (9):4991-5000 - 72.
Morrison DK. MAP kinase pathways. Cold Spring Harbor Perspectives in Biology. 2012; 4 (11). DOI: 10.1101/cshperspect.a011254 - 73.
Laubach JP, Fu P, Jiang X, Salter KH, Potti A, Arcasoy MO. Polycythemia vera erythroid precursors exhibit increased proliferation and apoptosis resistance associated with abnormal RAS and PI3K pathway activation. Experimental Hematology. 2009; 37 (12):1411-1422. DOI: 10.1016/j.exphem.2009.09.009 - 74.
Park S, Chapuis N, Tamburini J, Bardet V, Cornillet-Lefebvre P, Willems L, et al. Role of the PI3K/AKT and mTOR signaling pathways in acute myeloid leukemia. Haematologica. 2010; 95 (5):819-828. DOI: 10.3324/haematol.2009.013797 - 75.
Fransecky L, Mochmann LH, Baldus CD. Outlook on PI3K/AKT/mTOR inhibition in acute leukemia. Molecular and Cellular Therapies. 2015; 3 :2. DOI: 10.1186/s40591-015-0040-8 - 76.
Dai C, Chung IJ, Krantz SB. Increased erythropoiesis in polycythemia vera is associated with increased erythroid progenitor proliferation and increased phosphorylation of Akt/PKB. Experimental Hematology. 2005; 33 (2):152-158. DOI: 10.1016/j.exphem.2004.10.017 - 77.
Khan I, Huang Z, Wen Q , Stankiewicz MJ, Gilles L, Goldenson B, et al. AKT is a therapeutic target in myeloproliferative neoplasms. Leukemia. 2013; 27 (9):1882-1890. DOI: 10.1038/leu.2013.167 - 78.
Fu C, Wen QJ, Marinaccio C, Ling T, Chen W, Bulic M, et al. AKT activation is a feature of CALR mutant myeloproliferative neoplasms. Leukemia. 2019; 33 (1):271-274. DOI: 10.1038/s41375-018-0224-8 - 79.
Bartalucci N, Tozzi L, Bogani C, Martinelli S, Rotunno G, Villeval JL, et al. Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms. Journal of Cellular and Molecular Medicine. 2013; 17 (11):1385-1396. DOI: 10.1111/jcmm.12162 - 80.
Tefferi A. Myeloproliferative neoplasms: Thrombophilic clonal stem cell diseases. Cancer Treatment and Research. 2009; 148 :157-179. DOI: 10.1007/978-0-387-79962-9_10 - 81.
Scott LM. The JAK2 exon 12 mutations: A comprehensive review. American Journal of Hematology. 2011; 86 (8):668-676. DOI: 10.1002/ajh.22063 - 82.
Scott LM, Tong W, Levine RL, Scott MA, Beer PA, Stratton MR, et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. The New England Journal of Medicine. 2007; 356 (5):459-468. DOI: 10.1056/NEJMoa065202 - 83.
Pardanani A, Lasho TL, Finke C, Hanson CA, Tefferi A. Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera. Leukemia. 2007; 21 (9):1960-1963. DOI: 10.1038/sj.leu.2404810 - 84.
Butcher CM, Hahn U, To LB, Gecz J, Wilkins EJ, Scott HS, et al. Two novel JAK2 exon 12 mutations in JAK2V617F-negative polycythaemia vera patients. Leukemia. 2008; 22 (4):870-873. DOI: 10.1038/sj.leu.2404971 - 85.
Schnittger S, Bacher U, Haferlach C, Geer T, Muller P, Mittermuller J, et al. Detection of JAK2 exon 12 mutations in 15 patients with JAK2V617F negative polycythemia vera. Haematologica. 2009; 94 (3):414-418. DOI: 10.3324/haematol.13223 - 86.
Grinfeld J, Nangalia J, Green AR. Molecular determinants of pathogenesis and clinical phenotype in myeloproliferative neoplasms. Haematologica. 2017; 102 (1):7-17. DOI: 10.3324/haematol.2014.113845 - 87.
Staerk J, Lacout C, Sato T, Smith SO, Vainchenker W, Constantinescu SN. An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor. Blood. 2006; 107 (5):1864-1871. DOI:10.1182/blood-2005-06-2600 - 88.
Defour JP, Itaya M, Gryshkova V, Brett IC, Pecquet C, Sato T, et al. Tryptophan at the transmembrane-cytosolic junction modulates thrombopoietin receptor dimerization and activation. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110 (7):2540-2545. DOI: 10.1073/pnas.1211560110 - 89.
Chaligne R, Tonetti C, Besancenot R, Roy L, Marty C, Mossuz P, et al. New mutations of MPL in primitive myelofibrosis: Only the MPL W515 mutations promote a G1/S-phase transition. Leukemia. 2008; 22 (8):1557-1566. DOI: 10.1038/leu.2008.137 - 90.
Ding J, Komatsu H, Wakita A, Kato-Uranishi M, Ito M, Satoh A, et al. Familial essential thrombocythemia associated with a dominant-positive activating mutation of the c-MPL gene, which encodes for the receptor for thrombopoietin. Blood. 2004; 103 (11):4198-4200. DOI: 10.1182/blood-2003-10-3471 - 91.
Beer PA, Campbell PJ, Scott LM, Bench AJ, Erber WN, Bareford D, et al. MPL mutations in myeloproliferative disorders: Analysis of the PT-1 cohort. Blood. 2008; 112 (1):141-149. DOI: 10.1182/blood-2008-01-131664 - 92.
Milosevic Feenstra JD, Nivarthi H, Gisslinger H, Leroy E, Rumi E, Chachoua I, et al. Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms. Blood. 2016; 127 (3):325-332. DOI: 10.1182/blood-2015-07-661835 - 93.
Cabagnols X, Favale F, Pasquier F, Messaoudi K, Defour JP, Ianotto JC, et al. Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients. Blood. 2016; 127 (3):333-342. DOI: 10.1182/blood-2015-07-661983 - 94.
Cazzola M, Kralovics R. From Janus kinase 2 to calreticulin: The clinically relevant genomic landscape of myeloproliferative neoplasms. Blood. 2014; 123 (24):3714-3719. DOI: 10.1182/blood-2014-03-530865 - 95.
Chachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu RI, Marty C, et al. Thrombopoietin receptor activation by myeloproliferative neoplasm associated calreticulin mutants. Blood. 2016; 127 (10):1325-1335. DOI: 10.1182/blood-2015-11-681932 - 96.
Salati S, Genovese E, Prudente Z, Carretta C, Bartalucci N, Pennucci V, et al. Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in hematopoietic cells. American Society of Hematology. 2018 - 97.
Pettit K, Odenike O. Novel therapies for myelofibrosis. Current Hematologic Malignancy Reports. 2017; 12 (6):611-624. DOI: 10.1007/s11899-017-0403-0 - 98.
Tefferi A, Vannucchi AM, Barbui T. Polycythemia vera treatment algorithm 2018. Blood Cancer Journal. 2018; 8 (1):1-7. DOI:10.1038/s41408-017-0042-7 - 99.
Tefferi A, Barbui T. Polycythemia vera and essential thrombocythemia: 2019 update on diagnosis, risk-stratification and management. American Journal of Hematology. 2019; 94 (1):133-143. DOI: 10.1002/ajh.25303 - 100.
Marchioli R, Finazzi G, Specchia G, Cacciola R, Cavazzina R, Cilloni D, et al. Cardiovascular events and intensity of treatment in polycythemia vera. The New England Journal of Medicine. 2013; 368 (1):22-33. DOI: 10.1056/NEJMoa1208500 - 101.
Fleischman AG, Aichberger KJ, Luty SB, Bumm TG, Petersen CL, Doratotaj S, et al. TNFalpha facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms. Blood. 2011; 118 (24):6392-6398. DOI: 10.1182/blood-2011-04-348144 - 102.
Darding M, Feltham R, Tenev T, Bianchi K, Benetatos C, Silke J, et al. Molecular determinants of Smac mimetic induced degradation of cIAP1 and cIAP2. Cell Death and Differentiation. 2011; 18 (8):1376-1386. DOI: 10.1038/cdd.2011.10 - 103.
Welsh K, Milutinovic S, Ardecky RJ, Gonzalez-Lopez M, Ganji SR, Teriete P, et al. Characterization of potent SMAC mimetics that sensitize cancer cells to TNF family-induced apoptosis. PLoS One. 2016; 11 (9):e0161952:1-19. DOI: 10.1371/journal.pone.0161952 - 104.
Verger E, Soret-Dulphy J, Maslah N, Roy L, Rey J, Ghrieb Z, et al. Ropeginterferon alpha-2b targets JAK2V617F-positive polycythemia vera cells in vitro and in vivo. Blood Cancer Journal. 2018; 8 (10):94. DOI: 10.1038/s41408-018-0133-0 - 105.
Greenfield G, McPherson S, Mills K, McMullin MF. The ruxolitinib effect: Understanding how molecular pathogenesis and epigenetic dysregulation impact therapeutic efficacy in myeloproliferative neoplasms. Journal of Translational Medicine. 2018; 16 (1):360. DOI: 10.1186/s12967-018-1729-7 - 106.
Kiladjian JJ, Guglielmelli P, Griesshammer M, Saydam G, Masszi T, Durrant S, et al. Efficacy and safety of ruxolitinib after and versus interferon use in the RESPONSE studies. Annals of Hematology. 2018; 97 (4):617-627. DOI: 10.1007/s00277-017-3225-1 - 107.
Bose P, Verstovsek S. Developmental therapeutics in myeloproliferative neoplasms. Clinical Lymphoma, Myeloma & Leukemia. 2017; 17S :S43-S52. DOI: 10.1016/j.clml.2017.02.014 - 108.
Quintas-Cardama A, Vaddi K, Liu P, Manshouri T, Li J, Scherle PA, et al. Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms. Blood. 2010; 115 (15):3109-3117. DOI: 10.1182/blood-2009-04-214957 - 109.
Chase A, Bryant C, Score J, Haferlach C, Grossmann V, Schwaab J, et al. Ruxolitinib as potential targeted therapy for patients with JAK2 rearrangements. Haematologica. 2013; 98 (3):404-408. DOI: 10.3324/haematol.2012.067959 - 110.
Gozgit JM, Bebernitz G, Patil P, Ye M, Parmentier J, Wu J, et al. Effects of the JAK2 inhibitor, AZ960, on Pim/BAD/BCL-xL survival signaling in the human JAK2 V617F cell line SET-2. The Journal of Biological Chemistry. 2008; 283 (47):32334-32343. DOI: 10.1074/jbc.M803813200 - 111.
Harrison C, Vannucchi AM. Ruxolitinib: A potent and selective Janus kinase 1 and 2 inhibitor in patients with myelofibrosis. An update for clinicians. Therapeutic Advances in Hematology. 2012; 3 (6):341-354. DOI: 10.1177/2040620712459746 - 112.
Mazzacurati L, Lambert QT, Pradhan A, Griner LN, Huszar D, Reuther GW. The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells. Oncotarget. 2015; 6 (37):40141-40157. DOI: 10.18632/oncotarget.5653 - 113.
Spivak JL. Polycythemia vera: Myths, mechanisms, and management. Blood. 2002; 100 (13):4272-4290. DOI: 10.1182/blood-2001-12-0349 - 114.
Zeuner A, Pedini F, Francescangeli F, Signore M, Girelli G, Tafuri A, et al. Activity of the BH3 mimetic ABT-737 on polycythemia vera erythroid precursor cells. Blood. 2009; 113 (7):1522-1525. DOI: 10.1182/blood-2008-03-143321 - 115.
Will B, Siddiqi T, Jorda MA, Shimamura T, Luptakova K, Staber PB, et al. Apoptosis induced by JAK2 inhibition is mediated by Bim and enhanced by the BH3 mimetic ABT-737 in JAK2 mutant human erythroid cells. Blood. 2010; 115 (14):2901-2909. DOI: 10.1182/blood-2009-03-209544 - 116.
Winter PS, Sarosiek KA, Lin KH, Meggendorfer M, Schnittger S, Letai A, et al. RAS signaling promotes resistance to JAK inhibitors by suppressing BAD-mediated apoptosis. Science Signaling. 2014; 7 (357):ra122. DOI: 10.1126/scisignal.2005301 - 117.
Alonso-Alvarez S, Pardal E, Sanchez-Nieto D, Navarro M, Caballero MD, Mateos MV, et al. Plitidepsin: design, development, and potential place in therapy. Drug Design, Development and Therapy. 2017; 11 :253-264. DOI: 10.2147/DDDT.S94165 - 118.
Garcia-Fernandez LF, Losada A, Alcaide V, Alvarez AM, Cuadrado A, Gonzalez L, et al. Aplidin induces the mitochondrial apoptotic pathway via oxidative stress-mediated JNK and p38 activation and protein kinase C delta. Oncogene. 2002; 21 (49):7533-7544. DOI: 10.1038/sj.onc.1205972 - 119.
Munoz-Alonso MJ, Gonzalez-Santiago L, Martinez T, Losada A, Galmarini CM, Munoz A. The mechanism of action of plitidepsin. Current Opinion in Investigational Drugs. 2009; 10 (6):536-542 - 120.
Gonzalez-Santiago L, Suarez Y, Zarich N, Munoz-Alonso MJ, Cuadrado A, Martinez T, et al. Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation. Cell Death and Differentiation. 2006; 13 (11):1968-1981. DOI: 10.1038/sj.cdd.4401898 - 121.
Losada A, Lopez-Oliva JM, Sanchez-Puelles JM, Garcia-Fernandez LF. Establishment and characterisation of a human carcinoma cell line with acquired resistance to Aplidin. British Journal of Cancer. 2004; 91 (7):1405-1413. DOI: 10.1038/sj.bjc.6602166 - 122.
Pardanani A, Tefferi A, Guglielmelli P, Bogani C, Bartalucci N, Rodriguez J, et al. Evaluation of plitidepsin in patients with primary myelofibrosis and post polycythemia vera/essential thrombocythemia myelofibrosis: results of preclinical studies and a phase II clinical trial. Blood Cancer Journal. 2015; 5 :e286. DOI: 10.1038/bcj.2015.5 - 123.
Nguyen M, Cencic R, Ertel F, Bernier C, Pelletier J, Roulston A, et al. Obatoclax is a direct and potent antagonist of membrane-restricted Mcl-1 and is synthetic lethal with treatment that induces Bim. BMC Cancer. 2015; 15 :568. DOI: 10.1186/s12885-015-1582-5 - 124.
Hernandez-Ilizaliturri FJ, Khubchandani S, Olejniczak SH, Hosking P, Gruber E, Czuczman M. The BH3-mimetic obatoclax (GX15-070) posses a dual-mechanism of action and induces both apoptosis and autophagy-dependent cell death of B cell non-Hodgkin’s. Lymphoma (B-NHL) Cells. Blood. 2008; 112 (11):605; 50th ASH Annual Meeting 2008; San Francisco; 6-9 December 2008 - 125.
Stamelos VA, Fisher N, Bamrah H, Voisey C, Price JC, Farrell WE, et al. The BH3 mimetic obatoclax accumulates in lysosomes and causes their alkalinization. PLoS One. 2016; 11 (3):e0150696. DOI: 10.1371/journal.pone.0150696 - 126.
Parikh SA, Kantarjian H, Schimmer A, Walsh W, Asatiani E, El-Shami K, et al. Phase II study of obatoclax mesylate (GX15-070), a small-molecule BCL-2 family antagonist, for patients with myelofibrosis. Clinical Lymphoma, Myeloma & Leukemia. 2010; 10 (4):285-289. DOI: 10.3816/CLML.2010.n.059 - 127.
Patel AB, Vellore NA, Deininger MW. New strategies in myeloproliferative neoplasms: The evolving genetic and therapeutic landscape. Clinical Cancer Research. 2016; 22 (5):1037-1047. DOI: 10.1158/1078-0432.CCR-15-0905 - 128.
Pemmaraju N, Carter BZ, Kantarjian HM, Cortes JE, Kadia TM, Garcia-Manero G, et al. Results for phase II clinical trial of LCL161, a SMAC mimetic, in Patients with Primary Myelofibrosis (PMF), Post-Polycythemia Vera Myelofibrosis (post-PV MF) or Post-Essential Thrombocytosis Myelofibrosis (post-ET MF). Blood. 2016;128(22):3105; 58th ASH Annual Meeting 2016; San Diego; 3-6 December 2016