We previously demonstrated that resistance to hypoxia is a common feature of Haematopoietic Stem Cells (HSC) and Leukemic Stem Cells (LSC), but also that a hypoxia-selectable LSC subset is likely to exists within any type of leukemia, including stabilized clonal cells lines. In Chronic Myeloid Leukemia (CML), either cell lines or CD34+ cells explanted from CML patients, hypoxia completely suppresses the expression of BCR/Ablprotein but not that of BCR/Abl transcript, so that hypoxia-resistant cells, while remaining genetically leukemic, are independent of BCR/Abl signaling for their maintenance in vitro and resistant to treatment with Imatinib-Mesylate (IM). Thus, hypoxia-selectable LSC are refractory to IM due to the lack of its molecular target. This is very well in keeping with the notions that: (i) IM, despite its impressive efficacy as first-line therapy for patients with chronic-phase CML, induces a state of Minimal Residual Disease (MRD), rather than cure; (ii) LSC most likely sustain MRD and are thereby responsible for the late relapses of CML; (iii) tissue hypoxia provides optimal conditions for the homing of normal and neoplastic stem cells. The Extracellular signal-Regulated Kinase 5 (ERK5) is a Mitogen-Activated Protein Kinase involved in the control of cell survival and proliferation, as well as in the pathogenesis of a number of cancers, including CML. ERK5 is activated by cytokines regulating stem cell compartments and participates to cell response to hypoxia. The main targets of the experimental work of this thesis were: 1) to explore the role of ERK5 in the maintenance of leukemia stem and progenitor cells; 2) to address the effects of different pharmacological MEK5/ERK5 inhibitors on the survival and proliferation of CML cells, either bulk or LSC populations; 3) to gather information useful to identify innovative (combination) treatment capable to eliminate the IM-insensitive LSC and thereby MDR. The selection of leukemic progenitor cells (LPC)/LSC, as well as the effects of pharmacological ERK5 pathway inhibition on hypoxia-selected LPC/LSC, were estimated by the Culture-Repopulating Ability (CRA) assay, a non-clonal assay which measures the culture-repopulating power of normal or leukemic hematopoietic cells. On the basis of entity and kinetics of repopulation of secondary cultures (LC2) where cell growth is unrestrained, it is possible to estimate the LSC or LPC content of cell populations subjected to a selective treatment in primary cultures (LC1). In the experiments reported here, the CRA assay was used to estimate the content of hypoxia-resistant CML cell populations with LSC/LPC and to test the effects on these cell subsets of genetic or pharmacologic inhibition of the ERK5 pathway. The inhibitors used were the ERK5-specific inhibitor XMD8-92 and the MEK5-specific inhibitorsBIX02188 and BIX02189. Genetic inhibition of ERK5 was obtained using lentiviral vectors expressing ERK5-specific short hairpin RNAs. The effects of the inhibiting treatments were tested on the KCL22 and K562 human stabilized CML cell lines, where ERK5 is constitutively activated. The results obtained indicated that all three inhibitors were inactive on the bulk of CML cell population with respect to cell number in culture. On the other hand, XMD8-92 determined an appreciable increase of the percentage of KCL22, but not K562, cells in the G0/G1 phase of cell cycle and a decrease of the percentage of cells in S phase. BIX02188 and BIX02189 did not exhibit any appreciable effect on cell cycle phase distribution in either K562 or KCL22 cell line. XMD8-92, but not BIX02188 or BIX02189, also determined an increase of p27kip expression in both cell lines and reduced the basal apoptosis occurring in untreated K562 cell cultures. In hypoxia, where constitutive activation of ERK5 was suppressed, BIX02188 or BIX02189 did not affect the cell number significantly in either cell line. XMD8-92 treatment, on the contrary, resulted in a significant, although not marked, increase of viable cell number. A block of progression of CML cells to the S phase and the increase of p27Kip operated by XMD8-92 were observed in both cell lines. Consistently, neither BIX02188 nor BIX02189, which did not induce a detectable G0/G1 accumulation, increased the expression of p27Kip. Taken together with the data obtained in normoxia, these results point to a cytostatic, rather than cytotoxic, effect of ERK5-specific inhibition. Moreover, the treatment with XMD8-92, but not BIX02188 orBIX02189, resulted in a significant reduction of hypoxia-induced apoptosis. These results, together with those obtained in normoxia, suggest that the protective effect of XMD8-92 against basal apoptosis occurring in control cultures in normoxia is enhanced in hypoxia. The treatment of KCL22 and K562 cells with BIX02188 or BIX02189 in normoxic LC1 reduced the ability to repopulate LC2, while that with XMD8-92 almost completely suppressed LC2 repopulation. On the other hand, when hypoxic LC1 were treated with BIX02188 or BIX02189, LC2 repopulation was almost immediate. On the contrary, the treatment with XMD8-92 completely suppressed LC2 repopulation. These results indicate that MEK5 inhibitors have a modest effect on CRA of BCR/Ablprotein-expressing CML, actually accelerating BCR/Ablprotein re-expression in cells rescued from hypoxia. On the contrary, both BCR/Ablprotein-expressing and BCR/Ablprotein-negative subsets were markedly sensitive to the treatment with XMD8-92. Thus, XMD8-92 was inactive on the bulk of CML cell population, but capable to suppress completely the BCR/Ablprotein-positive LPC and the hypoxia-selected, BCR/Ablprotein-negative LPC/LSC. To confirm the above results while overcoming problems of interpretation of data due to possible off-target effects of ERK5-inhibiting drugs, K562 cells were infected with lentiviral vectors expressing shRNA against ERK5 (shERK5). In hypoxia, genetic knockdown of ERK5, unlike its pharmacological inhibition, impaired CML cell survival. These differences are likely due to the well-known property of ERK5 to regulate a number of genes by direct interaction, independently of its kinase activity. On the other hand, ERK5 knockdown suppressed LC2 repopulation driven by hypoxia-selected cells as much as the inhibition of ERK5 enzymatic activity by XMD8-92. Since we found that XMD8-92 does not inhibit the overall survival of CML cells, but suppresses hypoxia-selected LSC-like cell subsets, we tested the effects of the XMD8-92/IM combination. IM markedly reduced the number of viable cells in normoxia as well as in hypoxia. The combination with XMD8-92 determined a marginal, if any, enhancement of the inhibitory effect of IM. LC2 repopulation was reduced, but not abolished, by IM treatment of normoxic or hypoxic LC1. Importantly, XMD8-92, alone or in combination with IM, suppressed completely LC2 repopulation. The effectiveness of XMD8-92 demonstrated on hypoxia-selected cell subsets of stabilized CML lines was also tested on primary cells explanted from CML patients. In keeping with what observed for cell lines, XMD8-92, alone or in combination with IM, impaired culture repopulation driven by hypoxia-selected LSC from CML patients. These results indicate that the combined treatment of XMD8-92 with IM may be an useful approach to try to eradicate CML together with the induction of remission, being XMD8-92 active on LPC/LSC but not cell bulk, and, viceversa, IM very active on the bulk but unable to suppress CML progenitors.

Role of ERK5/BMK1 in the survival, proliferation and stemness of chronic myeloid leukemia cells in hypoxia / Ignazia Tusa. - (2014).

Role of ERK5/BMK1 in the survival, proliferation and stemness of chronic myeloid leukemia cells in hypoxia

TUSA, IGNAZIA
2014

Abstract

We previously demonstrated that resistance to hypoxia is a common feature of Haematopoietic Stem Cells (HSC) and Leukemic Stem Cells (LSC), but also that a hypoxia-selectable LSC subset is likely to exists within any type of leukemia, including stabilized clonal cells lines. In Chronic Myeloid Leukemia (CML), either cell lines or CD34+ cells explanted from CML patients, hypoxia completely suppresses the expression of BCR/Ablprotein but not that of BCR/Abl transcript, so that hypoxia-resistant cells, while remaining genetically leukemic, are independent of BCR/Abl signaling for their maintenance in vitro and resistant to treatment with Imatinib-Mesylate (IM). Thus, hypoxia-selectable LSC are refractory to IM due to the lack of its molecular target. This is very well in keeping with the notions that: (i) IM, despite its impressive efficacy as first-line therapy for patients with chronic-phase CML, induces a state of Minimal Residual Disease (MRD), rather than cure; (ii) LSC most likely sustain MRD and are thereby responsible for the late relapses of CML; (iii) tissue hypoxia provides optimal conditions for the homing of normal and neoplastic stem cells. The Extracellular signal-Regulated Kinase 5 (ERK5) is a Mitogen-Activated Protein Kinase involved in the control of cell survival and proliferation, as well as in the pathogenesis of a number of cancers, including CML. ERK5 is activated by cytokines regulating stem cell compartments and participates to cell response to hypoxia. The main targets of the experimental work of this thesis were: 1) to explore the role of ERK5 in the maintenance of leukemia stem and progenitor cells; 2) to address the effects of different pharmacological MEK5/ERK5 inhibitors on the survival and proliferation of CML cells, either bulk or LSC populations; 3) to gather information useful to identify innovative (combination) treatment capable to eliminate the IM-insensitive LSC and thereby MDR. The selection of leukemic progenitor cells (LPC)/LSC, as well as the effects of pharmacological ERK5 pathway inhibition on hypoxia-selected LPC/LSC, were estimated by the Culture-Repopulating Ability (CRA) assay, a non-clonal assay which measures the culture-repopulating power of normal or leukemic hematopoietic cells. On the basis of entity and kinetics of repopulation of secondary cultures (LC2) where cell growth is unrestrained, it is possible to estimate the LSC or LPC content of cell populations subjected to a selective treatment in primary cultures (LC1). In the experiments reported here, the CRA assay was used to estimate the content of hypoxia-resistant CML cell populations with LSC/LPC and to test the effects on these cell subsets of genetic or pharmacologic inhibition of the ERK5 pathway. The inhibitors used were the ERK5-specific inhibitor XMD8-92 and the MEK5-specific inhibitorsBIX02188 and BIX02189. Genetic inhibition of ERK5 was obtained using lentiviral vectors expressing ERK5-specific short hairpin RNAs. The effects of the inhibiting treatments were tested on the KCL22 and K562 human stabilized CML cell lines, where ERK5 is constitutively activated. The results obtained indicated that all three inhibitors were inactive on the bulk of CML cell population with respect to cell number in culture. On the other hand, XMD8-92 determined an appreciable increase of the percentage of KCL22, but not K562, cells in the G0/G1 phase of cell cycle and a decrease of the percentage of cells in S phase. BIX02188 and BIX02189 did not exhibit any appreciable effect on cell cycle phase distribution in either K562 or KCL22 cell line. XMD8-92, but not BIX02188 or BIX02189, also determined an increase of p27kip expression in both cell lines and reduced the basal apoptosis occurring in untreated K562 cell cultures. In hypoxia, where constitutive activation of ERK5 was suppressed, BIX02188 or BIX02189 did not affect the cell number significantly in either cell line. XMD8-92 treatment, on the contrary, resulted in a significant, although not marked, increase of viable cell number. A block of progression of CML cells to the S phase and the increase of p27Kip operated by XMD8-92 were observed in both cell lines. Consistently, neither BIX02188 nor BIX02189, which did not induce a detectable G0/G1 accumulation, increased the expression of p27Kip. Taken together with the data obtained in normoxia, these results point to a cytostatic, rather than cytotoxic, effect of ERK5-specific inhibition. Moreover, the treatment with XMD8-92, but not BIX02188 orBIX02189, resulted in a significant reduction of hypoxia-induced apoptosis. These results, together with those obtained in normoxia, suggest that the protective effect of XMD8-92 against basal apoptosis occurring in control cultures in normoxia is enhanced in hypoxia. The treatment of KCL22 and K562 cells with BIX02188 or BIX02189 in normoxic LC1 reduced the ability to repopulate LC2, while that with XMD8-92 almost completely suppressed LC2 repopulation. On the other hand, when hypoxic LC1 were treated with BIX02188 or BIX02189, LC2 repopulation was almost immediate. On the contrary, the treatment with XMD8-92 completely suppressed LC2 repopulation. These results indicate that MEK5 inhibitors have a modest effect on CRA of BCR/Ablprotein-expressing CML, actually accelerating BCR/Ablprotein re-expression in cells rescued from hypoxia. On the contrary, both BCR/Ablprotein-expressing and BCR/Ablprotein-negative subsets were markedly sensitive to the treatment with XMD8-92. Thus, XMD8-92 was inactive on the bulk of CML cell population, but capable to suppress completely the BCR/Ablprotein-positive LPC and the hypoxia-selected, BCR/Ablprotein-negative LPC/LSC. To confirm the above results while overcoming problems of interpretation of data due to possible off-target effects of ERK5-inhibiting drugs, K562 cells were infected with lentiviral vectors expressing shRNA against ERK5 (shERK5). In hypoxia, genetic knockdown of ERK5, unlike its pharmacological inhibition, impaired CML cell survival. These differences are likely due to the well-known property of ERK5 to regulate a number of genes by direct interaction, independently of its kinase activity. On the other hand, ERK5 knockdown suppressed LC2 repopulation driven by hypoxia-selected cells as much as the inhibition of ERK5 enzymatic activity by XMD8-92. Since we found that XMD8-92 does not inhibit the overall survival of CML cells, but suppresses hypoxia-selected LSC-like cell subsets, we tested the effects of the XMD8-92/IM combination. IM markedly reduced the number of viable cells in normoxia as well as in hypoxia. The combination with XMD8-92 determined a marginal, if any, enhancement of the inhibitory effect of IM. LC2 repopulation was reduced, but not abolished, by IM treatment of normoxic or hypoxic LC1. Importantly, XMD8-92, alone or in combination with IM, suppressed completely LC2 repopulation. The effectiveness of XMD8-92 demonstrated on hypoxia-selected cell subsets of stabilized CML lines was also tested on primary cells explanted from CML patients. In keeping with what observed for cell lines, XMD8-92, alone or in combination with IM, impaired culture repopulation driven by hypoxia-selected LSC from CML patients. These results indicate that the combined treatment of XMD8-92 with IM may be an useful approach to try to eradicate CML together with the induction of remission, being XMD8-92 active on LPC/LSC but not cell bulk, and, viceversa, IM very active on the bulk but unable to suppress CML progenitors.
2014
Persio Dello Sbarba
ITALIA
Ignazia Tusa
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