Contributions
Abstract: EP1068
Type: E-Poster Presentation
Session title: Myeloproliferative neoplasms - Biology & Translational Research
Background
Contrasting indolent forms of systemic mastocytosis (SM), advanced SM, including aggressive SM (ASM), mast cell leukemia (MCL), and SM with an associated hematopoietic neoplasm (SM-AHN) are characterized by SM-induced organ damage, disease progression and a poor prognosis. In most patients, KIT D816V is detected and is considered to act as a disease-driver. Despite encouraging results obtained with KIT D816V-targeting tyrosine kinase inhibitors (TKI), such as midostaurin, most patients fail to achieve a durable remission. This may be explained by additional oncogenic pathways. Therefore, current efforts are focusing on the identification of suitable combination partners for KIT D816V-targeting TKI. The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me) acts anti-neoplastic in various malignancies.
Aims
The aim of the present study was to evaluate the anti-neoplastic potential of CDDO-Me, alone or in combination with KIT D816V-targeting TKI, in neoplastic mast cells (MC).
Methods
Primary bone marrow (BM) cells were obtained from 15 patients with SM (indolent SM, n=3; ASM, n=2; MCL, n=3; SM-AHN, n=7). Furthermore, we employed the MCL-like cell lines HMC-1.1 (lacking KIT D816V), HMC-1.2 (KIT D816V+), ROSAKIT WT, ROSAKIT D816V and MCPV-1 (expressing RAS G12V, hTert and Large T). Proliferation was determined by measuring 3H-thymidine uptake. Apoptosis was quantified by flow cytometry. Western blot experiments were performed to determine drug effects on expression and phosphorylation of target proteins.
Results
CDDO-Me was found to suppress the proliferation of HMC-1, ROSA and MCPV-1 cells, with comparable IC50 values (0.1-0.5 µM). Interestingly, no differences were seen between cells lacking or expressing KIT D816V or RAS G12V. Growth arrest was accompanied by induction of apoptosis in all cell lines tested. In addition, CDDO-Me was found to inhibit the proliferation of primary neoplastic MC, with IC50-values ranging between 0.1 and 1 µM with no differences observed between SM variants and independent of previous treatment with midostaurin. Anti-neoplastic effects of CDDO-Me were accompanied by suppression of KIT and AKT expression in HMC-1 cells. However, CDDO-Me also led to upregulation of heme oxygenase-1 (HO-1) in MC lines. Therefore, we combined CDDO-Me with the HO-1 inhibitor SMA-ZnPP. This combination resulted in synergistic effects in all cell lines tested. Finally, potential cooperative effects between CDDO-Me and the KIT D816V-targeting TKI midostaurin, avapritinib and nintedanib, were explored. In these experiments, CDDO-Me was found to synergize with KIT-targeting drugs in producing growth-inhibition in HMC-1 cells as well as in the multi drug-resistant cell line MCPV-1.
Conclusion
Together, we found that CDDO-Me counteracts growth and survival of various MCL-like cell lines and primary neoplastic MC in SM. CDDO-Me was found to potentiate anti-neoplastic effects of KIT D816V-targeting TKI and to synergize with the HO-1 blocker SMA-ZnPP in producing growth inhibition. Whether CDDO-Me is also effective in vivo in patients with ASM/MCL remains at present unknown.
Keyword(s): Mastocytosis
Abstract: EP1068
Type: E-Poster Presentation
Session title: Myeloproliferative neoplasms - Biology & Translational Research
Background
Contrasting indolent forms of systemic mastocytosis (SM), advanced SM, including aggressive SM (ASM), mast cell leukemia (MCL), and SM with an associated hematopoietic neoplasm (SM-AHN) are characterized by SM-induced organ damage, disease progression and a poor prognosis. In most patients, KIT D816V is detected and is considered to act as a disease-driver. Despite encouraging results obtained with KIT D816V-targeting tyrosine kinase inhibitors (TKI), such as midostaurin, most patients fail to achieve a durable remission. This may be explained by additional oncogenic pathways. Therefore, current efforts are focusing on the identification of suitable combination partners for KIT D816V-targeting TKI. The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me) acts anti-neoplastic in various malignancies.
Aims
The aim of the present study was to evaluate the anti-neoplastic potential of CDDO-Me, alone or in combination with KIT D816V-targeting TKI, in neoplastic mast cells (MC).
Methods
Primary bone marrow (BM) cells were obtained from 15 patients with SM (indolent SM, n=3; ASM, n=2; MCL, n=3; SM-AHN, n=7). Furthermore, we employed the MCL-like cell lines HMC-1.1 (lacking KIT D816V), HMC-1.2 (KIT D816V+), ROSAKIT WT, ROSAKIT D816V and MCPV-1 (expressing RAS G12V, hTert and Large T). Proliferation was determined by measuring 3H-thymidine uptake. Apoptosis was quantified by flow cytometry. Western blot experiments were performed to determine drug effects on expression and phosphorylation of target proteins.
Results
CDDO-Me was found to suppress the proliferation of HMC-1, ROSA and MCPV-1 cells, with comparable IC50 values (0.1-0.5 µM). Interestingly, no differences were seen between cells lacking or expressing KIT D816V or RAS G12V. Growth arrest was accompanied by induction of apoptosis in all cell lines tested. In addition, CDDO-Me was found to inhibit the proliferation of primary neoplastic MC, with IC50-values ranging between 0.1 and 1 µM with no differences observed between SM variants and independent of previous treatment with midostaurin. Anti-neoplastic effects of CDDO-Me were accompanied by suppression of KIT and AKT expression in HMC-1 cells. However, CDDO-Me also led to upregulation of heme oxygenase-1 (HO-1) in MC lines. Therefore, we combined CDDO-Me with the HO-1 inhibitor SMA-ZnPP. This combination resulted in synergistic effects in all cell lines tested. Finally, potential cooperative effects between CDDO-Me and the KIT D816V-targeting TKI midostaurin, avapritinib and nintedanib, were explored. In these experiments, CDDO-Me was found to synergize with KIT-targeting drugs in producing growth-inhibition in HMC-1 cells as well as in the multi drug-resistant cell line MCPV-1.
Conclusion
Together, we found that CDDO-Me counteracts growth and survival of various MCL-like cell lines and primary neoplastic MC in SM. CDDO-Me was found to potentiate anti-neoplastic effects of KIT D816V-targeting TKI and to synergize with the HO-1 blocker SMA-ZnPP in producing growth inhibition. Whether CDDO-Me is also effective in vivo in patients with ASM/MCL remains at present unknown.
Keyword(s): Mastocytosis