Ignazia Tusa

The metabolically-modulated stem cell niche: a dynamic scenario regulating cancer cell phenotype and resistance to therapy.

This Perspective addresses the interactions of cancer stem cells (CSC) with environment which result in the modulation of CSC metabolism, and thereby of CSC phenotype and resistance to therapy. We considered first as a model disease chronic myeloid leukemia (CML), which is triggered by a well-identified oncogenetic protein (BCR/Abl) and brilliantly treated with tyrosine kinase inhibitors (TKi). However, TKi are extremely effective in inducing remission of disease, but unable, in most cases, to prevent relapse. We demonstrated that the interference with cell metabolism (oxygen/glucose shortage) enriches cells exhibiting the leukemia stem cell (LSC) phenotype and, at the same time, suppresses BCR/Abl protein expression. These LSC are therefore refractory to the TKi Imatinib-mesylate, pointing to cell metabolism as an important factor controlling the onset of TKi-resistant minimal residual disease (MRD) of CML and the related relapse. Studies of solid neoplasias brought another player into the control of MRD, low tissue pH, which often parallels cancer growth and progression. Thus, a 3-party scenario emerged for the regulation of CSC/LSC maintenance, MRD induction and disease relapse: the “hypoxic” versus the “ischemic” vs. the “acidic” environment. As these environments are unlikely constrained within rigid borders, we named this model the “metabolically-modulated stem cell niche.”

The mitogen-activated protein kinase ERK5 regulates the development and growth of hepatocellular carcinoma

Objective The extracellular signal-regulated kinase 5 (ERK5 or BMK1) is involved in tumour development. The ERK5 gene may be amplified in hepatocellular carcinoma (HCC), but its biological role has not been clarified. In this study, we explored the role of ERK5 expression and activity in HCC in vitro and in vivo. Design ERK5 expression was evaluated in human liver tissue. Cultured HepG2 and Huh-7 were studied after ERK5 knockdown by siRNA or in the presence of the specific pharmacological inhibitor, XMD8-92. The role of ERK5 in vivo was assessed using mouse Huh-7 xenografts. Results In tissue specimens from patients with HCC, a higher percentage of cells with nuclear ERK5 expression was found both in HCC and in the surrounding cirrhotic tissue compared with normal liver tissue. Inhibition of ERK5 decreased HCC cell proliferation and increased the proportion of cells in G0/G1 phase. These effects were associated with increased expression of p27 and p15 and decreased CCND1. Treatment with XMD8-92 or ERK5 silencing prevented cell migration induced by epidermal growth factor or hypoxia and caused cytoskeletal remodelling. In mouse xenografts, the rate of tumour appearance and the size of tumours were significantly lower when Huh-7 was silenced for ERK5. Moreover, systemic treatment with XMD8-92 of mice with established HCC xenografts markedly reduced tumour growth and decreased the expression of the proto-oncogene c-Rel. Conclusions ERK5 regulates the biology of HCC cells and modulates tumour development and growth in vivo. This pathway should be investigated as a possible therapeutic target in HCC.

AML1/ETO sensitizes via TRAIL acute myeloid leukemia cells to the pro-apoptotic effects of hypoxia.

We determined the effects of severe hypoxia (∼0.1% O2) on acute myeloid leukemia cells expressing the AML1/ETO oncogene. Incubation of Kasumi-1 cells in hypoxia induced growth arrest, apoptosis and reduction of AML1/ETO protein expression. The conditional expression of AML1/ETO in U937-A/E cells showed that hypoxia induces marked apoptosis in AML1/ETO-expressing cells only, pointing to AML1/ETO as a factor predisposing cells to hypoxia-induced apoptosis. In AML1/ETO-expressing cells, hypoxia enhanced TRAIL expression and its proapoptotic effects. AML1/ETO was found to bind TRAIL promoter and induce TRAIL transcription, although TRAIL expression was restrained by a concomitant relative transcription block. In hypoxia, such a TRAIL repression was removed and an increase of TRAIL expression was induced. Finally, blocking anti-TRAIL antibodies markedly reduced (Kasumi-1 cells) or completely inhibited (U937-A/E cells) hypoxia-induced apoptosis. Taken together, these results indicated that hypoxia induces apoptosis in AML1/ETO-expressing cells via a TRAIL/caspase 8-dependent autocrine loop and that TRAIL is a key regulator of hypoxia-induced apoptosis in these cells.

Targeting chronic myeloid leukemia stem cells with the hypoxia-inducible factor inhibitor acriflavine

Chronic myeloid leukemia (CML) is a hematopoietic stem cell (HSC)-driven neoplasia characterized by expression of the constitutively active tyrosine kinase BCR/Abl. CML therapy based on tyrosine kinase inhibitors (TKIs) is highly effective in inducing remission but not in targeting leukemia stem cells (LSCs), which sustain minimal residual disease and are responsible for CML relapse following discontinuation of treatment. The identification of molecules capable of targeting LSCs appears therefore of primary importance to aim at CML eradication. LSCs home in bone marrow areas at low oxygen tension, where HSCs are physiologically hosted. This study addresses the effects of pharmacological inhibition of hypoxia-inducible factor-1 (HIF-1), a critical regulator of LSC survival, on the maintenance of CML stem cell potential. We found that the HIF-1 inhibitor acriflavine (ACF) decreased survival and growth of CML cells. These effects were paralleled by decreased expression of c-Myc and stemness-related genes. Using different in vitro stem cell assays, we showed that ACF, but not TKIs, targets the stem cell potential of CML cells, including primary cells explanted from 12 CML patients. Moreover, in a murine CML model, ACF decreased leukemia development and reduced LSC maintenance. Importantly, ACF exhibited significantly less-severe effects on non-CML hematopoietic cells in vitro and in vivo. Thus, we propose ACF, a US Food and Drug Administration (FDA)-approved drug for nononcological use in humans, as a novel therapeutic approach to prevent CML relapse and, in combination with TKIs, enhance induction of remission.

ERK5 is activated by oncogenic BRAF and promotes melanoma growth

Malignant melanoma is among the most aggressive cancers and its incidence is increasing worldwide. Targeted therapies and immunotherapy have improved the survival of patients with metastatic melanoma in the last few years; however, available treatments are still unsatisfactory. While the role of the BRAF-MEK1/2-ERK1/2 pathway in melanoma is well established, the involvement of mitogen-activated protein kinases MEK5-ERK5 remains poorly explored. Here we investigated the function of ERK5 signaling in melanoma. We show that ERK5 is consistently expressed in human melanoma tissues and is active in melanoma cells. Genetic silencing and pharmacological inhibition of ERK5 pathway drastically reduce the growth of melanoma cells and xenografts harboring wild-type (wt) or mutated BRAF (V600E). We also found that oncogenic BRAF positively regulates expression, phosphorylation, and nuclear localization of ERK5. Importantly, ERK5 kinase and transcriptional transactivator activities are enhanced by BRAF. Nevertheless, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Accordingly, combination of MEK5 or ERK5 inhibitors with BRAFV600E inhibitor vemurafenib is more effective than single treatments in reducing colony formation and growth of BRAFV600E melanoma cells and xenografts. Overall, these data support a key role of the ERK5 pathway for melanoma growth in vitro and in vivo and suggest that targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, might represent a novel approach for melanoma treatment.

Salarin C inhibits the maintenance of chronic myeloid leukemia progenitor cells

We previously showed that incubation of chronic myeloid leukemia (CML) cells in very low oxygen selects a cell subset where the oncogenetic BCR/Abl protein is suppressed and which is thereby refractory to tyrosine kinase inhibitors used for CML therapy. In this study, salarin C, an anticancer macrolide extracted from the Fascaplysinopsis sponge, was tested as for its activity on CML cells, especially after their incubation in atmosphere at 0.1% oxygen. Salarin C induced mitotic cycle arrest, apoptosis and DNA damage. Salarin C also concentration-dependently inhibited the maintenance of stem cell potential in cultures in low oxygen of either CML cell lines or primary cells. Surprisingly, the drug also concentration-dependently enforced the maintenance of BCR/Abl signaling in low oxygen, an effect which was paralleled by the rescue of sensitivity of stem cell potential to IM. These results suggest a potential use of salarin C for the suppression of CML cells refractory to tyrosine kinase inhibitors

Targeting the Extracellular Signal-Regulated Kinase 5 Pathway to Suppress Human Chronic Myeloid Leukemia Stem Cells

Summary Tyrosine kinase inhibitors (TKi) are effective against chronic myeloid leukemia (CML), but their inefficacy on leukemia stem cells (LSCs) may lead to relapse. To identify new druggable targets alternative to BCR/ABL, we investigated the role of the MEK5/ERK5 pathway in LSC maintenance in low oxygen, a feature of bone marrow stem cell niches. We found that MEK5/ERK5 pathway inhibition reduced the growth of CML patient-derived cells and cell lines in vitro and the number of leukemic cells in vivo. Treatment in vitro of primary CML cells with MEK5/ERK5 inhibitors, but not TKi, strikingly reduced culture repopulation ability (CRA), serial colony formation ability, long-term culture-initiating cells (LTC-ICs), and CD26-expressing cells. Importantly, MEK5/ERK5 inhibition was effective on CML cells regardless of the presence or absence of imatinib, and did not reduce CRA or LTC-ICs of normal CD34+ cells. Thus, targeting MEK/ERK5 may represent an innovative therapeutic approach to suppress CML progenitor/stem cells.

PO-099 Targeting the mitogen activated protein kinase ERK5 in human melanoma

Introduction Melanoma is the most aggressive skin cancer with a poor prognosis in advanced stages. Available treatments for melanoma are unsatisfactory, because rapidly lead to an acquired resistance in the majority of cases. Therefore, there is urgent need to identify novel possible targets involved in melanoma growth. ERK5/BMK1 is a member of the Mitogen-Activated Protein Kinases (MAPK) family and regulates cell functions critical for tumour development. Indeed, several studies reported a direct involvement of ERK5 in several types of cancer including prostate and breast cancer and hepatocellular carcinoma. However, no data have been reported about a possible role of ERK5 in melanoma. Material and methods Cell lines and patient-derived primary melanoma cells (wild type B-RAF: SSM2c and M26c; BRAFV600E: A375, SK-Mel-5, SK-Mel-28, 501-Mel, expressing; NRASQ61R: SK-Mel-2; MeWo) have been used for in vitro and in vivo experiments. HEK293T cells were used for protein overexpression. ERK5 inhibition was achieved using ERK5 and MEK5 inhibitors or lentiviral vectors encoding shRNA specific for ERK5. BRAF inhibition was achieved using Vemurafenib, a BRAFV600E inhibitor. Results and discussions In silico data analysis indicated that components of the ERK5 pathway are upregulated in up to 47% melanoma patients. Accordingly, we found that ERK5 is consistently expressed and active in commercial and patients derived melanoma cell lines. On that basis, we investigated the role of ERK5 in melanoma cell growth. In vitro, pharmacological or genetic inhibition of ERK5 decreased the number of viable cells in several melanoma cell lines. Moreover, xenografts performed using LV-shERK5-transduced A375 or SSM2c cells showed a reduced tumour growth when compared to those transduced with control LV-shC. We also found that oncogenic BRAF positively regulates expression, phosphorylation and nuclear localization of exogenous and endogenous ERK5. Accordingly, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Furthermore, the combination of MEK5 or ERK5 inhibitors with vemurafenib is more effective than single treatments in reducing 2D colony formation and growth of BRAFV600E melanoma cells and xenografts. Conclusion Our results identify ERK5 as a critical regulator of melanoma growth in vitro and in vivo, and point toward the possibility of targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, for the treatment of melanoma.

PO-145 ERK5 pathway inhibitors inhibit the maintenance of chronic myeloid leukaemia stem cells

Introduction Chronic myeloid leukaemia (CML) is a hematopoietic stem cell (HSC)-driven neoplasia characterised by the expression of the constitutively active tyrosine kinase BCR/ABL. CML therapy based on tyrosine kinase inhibitors (TKi) is highly effective in inducing remission but not in targeting leukaemia stem cells (LSC), which sustain the minimal residual disease and are responsible for CML relapse following discontinuation of treatment. Our aim was to address the effects of the inhibition of the ERK5 pathway on the maintenance of CML LSC. Material and methods KCL22 and K562 CML cell lines, patient-derived CML cells or CD34 +peripheral blood cells from healthy donors (informed consent) were incubated in normoxic or hypoxic (0.1% O2) primary cultures (LC1) in the presence or the absence of drugs. At the end of incubation (day 7), cells were analysed on a flow cytometer to determine the expression of stem cell markers or transferred to drug-free normoxic secondary cultures (LC2) to measure LC2 repopulation as a read-out of progenitor/stem cell potential (CRA assay). In the serial Colony Formation Ability (CFA) assay colonies were scored on day 7 of each passage (III passages). In the Long-Term Culture-Initiating Cells (LTC-IC) assay the number of colonies was scored after 14 days. Compounds: XMD8-92 (ERK5 inhibitor) and BIX02189 (MEK5 inhibitor); imatinib and dasatinib (BCR/ABL inhibitors). Results and discussions In CML patient-derived cells and cell lines, we found that the MEK5/ERK5 pathway is active and necessary for optimal proliferation in low oxygen, a condition typical of normal hematopoietic and leukemic stem cell niches. Treatment of primary CML cells with XMD8-92 or BIX02189, but not with TKi, strikingly reduced Culture Repopulation Ability (CRA), serial Colony Formation Ability and Long-Term Culture-Initiating Cells (LTC-IC). Importantly, inhibition of MEK5/ERK5 was effective on CML cells regardless of the presence or absence of imatinib (IM), and did not reduce CRA or LTC-IC of normal CD34 +cells. Interestingly, in hypoxia, combined treatment XMD8-92/IM decreased the expression of genes relevant for stem cell maintenance such as c-MYC, SOX2 and NANOG and the expression of CD26, a CML LSC marker. Conclusion We propose ERK5 pathway inhibitors as a novel therapeutic approach to prevent CML relapse and, in combination with TKi, enhance induction of remission.

Abstract 3904: Inhibition of the ERK5 pathway as a novel approach to target human chronic myeloid leukemia stem cells

Tyrosine kinase inhibitors (TKi) targeting BCR/ABL are very effective for the treatment of Chronic Myeloid Leukaemia (CML). However, resistance mechanisms or their inefficacy on CML leukaemia stem cells (LSC) may lead to relapse. Therefore, there is urgent need to identify new molecular targets. The Extracellular signal-Regulated Kinase 5 (ERK5) is a Mitogen-Activated Protein Kinase involved in cancer. Our aim was to study a possible role of the ERK5 pathway in CML LSC. Cells used were: human CML cell lines K562 and KCL22 that express constitutively active ERK5; CML patient-derived cells and CD34+ cells from heathy donors (after informed consent had been obtained). Cells were incubated in normoxic (routine) or hypoxic (0.2% O2) primary cultures (LC1) in the presence or the absence of drugs. Day-7 LC1 cells were transferred to drug-free, non-selective normoxic secondary cultures (LC2), to measure LC2 repopulation as a read-out of progenitor/stem cell potential (CRA assay). Compounds: XMD8-92 (ERK5 inhibitor) and BIX02189 (MEK5 inhibitor); imatinib (BCR/ABL inhibitor, IM). We previously showed that stem cell potential of CML LSC is maintained in severe hypoxia. In K562 and KCL22 cells and in primary cells derived from 9 CML patients, the treatment in hypoxic LC1 with XMD8-92 or BIX02189, but not with IM, impaired progenitor/stem cell potential. The same results were obtained by combined treatment of XMD892 with IM. Importantly, XMD8-92 did not affect progenitor/stem cell potential of CD34+ cells from heathy donors. In colony formation ability assays ERK5 inhibition decreased colony formation by human primary CML cells to a higher extent than that by normal human CD34+ hematopoietic cells. Interestingly, in hypoxia, combined treatment XMD8-92/IM decreased the expression of genes relevant for stem cell maintenance such as p21, nanog and c-myc and the expression of CD26, a CML LSC marker. Moreover, combined XMD8- 92/IM maintained low the expression of p27, another gene involved in stem cell maintenance, that is increased by either drug when administered alone. This indicate that the ERK5 pathway inhibitors impaired LSC maintenance of CML cell lines and primary CML cells. Citation Format: Ignazia Tusa, Giulia Cheloni, Nathanael Gray, Antonella Gozzini, Persio Dello Sbarba, Elisabetta Rovida. Inhibition of the ERK5 pathway as a novel approach to target human chronic myeloid leukemia stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3904. doi:10.1158/1538-7445.AM2017-3904