Contributions
Abstract: EP1065
Type: E-Poster Presentation
Session title: Myeloproliferative neoplasms - Biology & Translational Research
Background
Advanced systemic mastocytosis (AdvSM), including aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN) and mast cell leukemia (MCL) are rare, KIT-driven malignancies with a grave prognosis. Although KIT D816V inhibitors, such as midostaurin or avapritinib, are available, most patients do not achieve a durable complete remission upon treatment with such drugs. Drug combinations may be an approach to improve the outcome of these patients. Cyclin-dependent kinase-4 (CDK4) and CDK6 contribute to cell cycle initiation and growth of normal and neoplastic cells. However, CDK4/CDK6 have not been analyzed as targets in AdvSM so far.
Aims
We aimed at elucidating the role of CDK4/CDK6 as therapeutic targets in AdvSM and evaluated anti-proliferative effects of CDK4/CDK6-inhibitors, alone or in combination with KIT D816V-targeting drugs, on neoplastic mast cells (MC).
Methods
Primary neoplastic cells were isolated from bone marrow (BM) samples of 21 patients [indolent SM (ISM; n=5), ASM (n=2), MCL (n=3) and SM-AHN (n=11)] and used in proliferation studies. mRNA was obtained from 70 additional patients with SM [ISM (n=45), SSM (n=2), ASM (n=7), SM-AHND (n=11), MCL (n=5)]. 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) were also tested. Expression of CDK4, CDK6 and cyclins D1 and D2 was analyzed by qRT-PCR. Phosphorylation of the retinoblastoma gene product (RB1) was analyzed by Western blotting. The functional role of CDKs was evaluated by shRNA-mediated knockdown of CDK4 and/or CDK6 in HMC-1.2 cells. Proliferation was determined by measuring 3H-thymidine uptake. Apoptosis was quantified by flow cytometry.
Results
CDK4, CDK6 and cyclins D1 and D2 were found to be overexpressed in BM samples from patients with AdvSM compared to samples of ISM patients. shRNA-mediated knockdown of both CDK4 and CDK6 resulted in a growth-arrest in HMC-1.2 cells, suggesting a functional role of CDK4/CDK6 in MC expansion. The pharmacologic CDK4/CDK6-inhibitors palbociclib, ribociclib and abemaciclib were found to suppress proliferation of HMC-1 cells and ROSA cells expressing or lacking KIT D816V, with reasonable IC50 values (<0.5 µM). In MCPV-1 cells, abemaciclib also induced growth-inhibition (IC50: 1–3 µM), whereas no substantial growth-inhibitory effects were seen with palbociclib and ribociclib (IC50 >10 µM). However, neoplastic cells in all primary samples tested were found to be sensitive to palbociclib, ribociclib and abemaciclib (IC50: <0.5 µM), with no differences seen between sub-types of SM. In the MCL-like cell lines, growth-inhibition was accompanied by apoptosis, cell cycle arrest and inhibition of phospho-RB1, the main target of CDK4/CDK6. CDK4/CDK6 inhibitors were also found to induce apoptosis in CD34+/CD38− stem cells obtained from 3 patients with AdvSM. Finally, cooperative effects of drug combinations involving CDK4/CDK6-inhibitors and KIT D816V-targeting drugs were assessed. In these experiments, palbociclib, ribociclib and abemaciclib were found to synergize with the KIT-inhibitors midostaurin, avapritinib and nintedanib in inducing growth inhibition in all MCL-like cell lines, including MCPV-1 as well as in primary neoplastic MC.
Conclusion
CDK4/CDK6 inhibition is a potent approach to block growth and survival of neoplastic MC and to potentiate anti-proliferative effects of KIT-targeting drugs in AdvSM. Whether such concepts can improve clinical outcomes in patients with drug-resistant AdvSM remains to be determined in clinical trials.
Keyword(s): Cell cycle progression, Kit, Mastocytosis, Targeted therapy
Abstract: EP1065
Type: E-Poster Presentation
Session title: Myeloproliferative neoplasms - Biology & Translational Research
Background
Advanced systemic mastocytosis (AdvSM), including aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN) and mast cell leukemia (MCL) are rare, KIT-driven malignancies with a grave prognosis. Although KIT D816V inhibitors, such as midostaurin or avapritinib, are available, most patients do not achieve a durable complete remission upon treatment with such drugs. Drug combinations may be an approach to improve the outcome of these patients. Cyclin-dependent kinase-4 (CDK4) and CDK6 contribute to cell cycle initiation and growth of normal and neoplastic cells. However, CDK4/CDK6 have not been analyzed as targets in AdvSM so far.
Aims
We aimed at elucidating the role of CDK4/CDK6 as therapeutic targets in AdvSM and evaluated anti-proliferative effects of CDK4/CDK6-inhibitors, alone or in combination with KIT D816V-targeting drugs, on neoplastic mast cells (MC).
Methods
Primary neoplastic cells were isolated from bone marrow (BM) samples of 21 patients [indolent SM (ISM; n=5), ASM (n=2), MCL (n=3) and SM-AHN (n=11)] and used in proliferation studies. mRNA was obtained from 70 additional patients with SM [ISM (n=45), SSM (n=2), ASM (n=7), SM-AHND (n=11), MCL (n=5)]. 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) were also tested. Expression of CDK4, CDK6 and cyclins D1 and D2 was analyzed by qRT-PCR. Phosphorylation of the retinoblastoma gene product (RB1) was analyzed by Western blotting. The functional role of CDKs was evaluated by shRNA-mediated knockdown of CDK4 and/or CDK6 in HMC-1.2 cells. Proliferation was determined by measuring 3H-thymidine uptake. Apoptosis was quantified by flow cytometry.
Results
CDK4, CDK6 and cyclins D1 and D2 were found to be overexpressed in BM samples from patients with AdvSM compared to samples of ISM patients. shRNA-mediated knockdown of both CDK4 and CDK6 resulted in a growth-arrest in HMC-1.2 cells, suggesting a functional role of CDK4/CDK6 in MC expansion. The pharmacologic CDK4/CDK6-inhibitors palbociclib, ribociclib and abemaciclib were found to suppress proliferation of HMC-1 cells and ROSA cells expressing or lacking KIT D816V, with reasonable IC50 values (<0.5 µM). In MCPV-1 cells, abemaciclib also induced growth-inhibition (IC50: 1–3 µM), whereas no substantial growth-inhibitory effects were seen with palbociclib and ribociclib (IC50 >10 µM). However, neoplastic cells in all primary samples tested were found to be sensitive to palbociclib, ribociclib and abemaciclib (IC50: <0.5 µM), with no differences seen between sub-types of SM. In the MCL-like cell lines, growth-inhibition was accompanied by apoptosis, cell cycle arrest and inhibition of phospho-RB1, the main target of CDK4/CDK6. CDK4/CDK6 inhibitors were also found to induce apoptosis in CD34+/CD38− stem cells obtained from 3 patients with AdvSM. Finally, cooperative effects of drug combinations involving CDK4/CDK6-inhibitors and KIT D816V-targeting drugs were assessed. In these experiments, palbociclib, ribociclib and abemaciclib were found to synergize with the KIT-inhibitors midostaurin, avapritinib and nintedanib in inducing growth inhibition in all MCL-like cell lines, including MCPV-1 as well as in primary neoplastic MC.
Conclusion
CDK4/CDK6 inhibition is a potent approach to block growth and survival of neoplastic MC and to potentiate anti-proliferative effects of KIT-targeting drugs in AdvSM. Whether such concepts can improve clinical outcomes in patients with drug-resistant AdvSM remains to be determined in clinical trials.
Keyword(s): Cell cycle progression, Kit, Mastocytosis, Targeted therapy