Contributions
Abstract: EP902
Type: E-Poster Presentation
Session title: Myelodysplastic syndromes - Biology & Translational Research
Background
In chronic myelomonocytic leukemia (CMML), therapeutic options include hematopoietic stem cell transplantation, chemotherapy, demethylating agents and hydroxyurea (HU), which is used for palliative cytoreduction. Despite these approaches, progression to secondary acute myeloid leukemia (sAML) is frequently seen and the prognosis is poor. No targeted therapies are available in CMML. CDK4 and CDK6 serve as major drug targets in applied oncology. However, the effects of CDK4/CDK6 inhibitors have not been analyzed in CMML contexts so far.
Aims
We examined the anti-neoplastic effects of HU, which has been described to counteract CDK4/CDK6-expression in leukemic cells, and the specific CDK4/CDK6 inhibitors palbociclib, ribociclib and abemaciclib on primary CMML cells and various monoblastic cell lines. We also aimed to define cooperative anti-neoplastic effects of CDK4/CDK6-targeting drugs and the multi-kinase inhibitor ponatinib.
Methods
Primary leukemic cells of the bone marrow were obtained from 11 patients: 8 with CMML, 3 with sAML following CMML. We also analyzed the monoblastic cell lines THP-1, MONO-MAC-6 (MM6) and U937. Cell proliferation was determined by 3H-thymidine uptake. Apoptosis and cell cycle distribution were quantified by flow cytometry. CDK4 and CDK6 mRNA were quantified by qPCR and protein expression and phosphorylation by Western blot analysis.
Results
Both HU and the CDK4/CDK6 inhibitors tested were found to disrupt the CDK4/CDK6-pathway in THP-1 and MM6 cells. Whereas HU decreased CDK4 and CDK6 mRNA- and protein expression, palbociclib, ribociclib and abemaciclib suppressed the expression and phosphorylation of RB1, the main target of CDK4/CDK6. HU was found to inhibit proliferation in all cell lines (IC50: THP-1: 25-50 µM; MM6: 100-150 µM; U937: 500-750 µM) and in all primary cell samples tested (IC50: 50-250 µM). Moreover, the CDK4/CDK6 inhibitors palbociclib, ribociclib and abemaciclib blocked cell proliferation in all cell lines (IC50: 0.5–10 µM) and all primary cell samples examined (IC50: 0.01–0.5 µM). Anti-proliferative effects were accompanied by cell cycle arrest and induction of apoptosis. We next searched for suitable combination partners for CDK4/CDK6 inhibitors and identified ponatinib, a broad-spectrum multi-kinase inhibitor. Ponatinib was found to inhibit proliferation in all monoblastic cell lines (IC50: 0.05-2.5 µM) and in primary leukemic cells (IC50: 0.01-2.5 µM). The drug combinations HU+ponatinib, palbociclib+ponatinib, ribociclib+ponatinib and abemaciclib+ponatinib resulted in strong synergistic anti-proliferative effects in THP-1, MM6 and primary cells isolated from 2 CMML patients. Interestingly, the combinations HU+palbociclib, HU+ribociclib, and HU+abemaciclib also resulted in synergistic growth inhibition, a phenomenon, that can be best explained by different drug target interaction profiles. Finally, we were able to demonstrate, that the triple combination `HU+palbociclib+ponatinib´ exerts superior growth-inhibitory effects in THP-1 cells compared to all 2-drug combinations tested.
Conclusion
HU and specific CDK4/CDK6-targeting drugs inhibit the proliferation of CMML cells and synergize with ponatinib in producing growth-arrest. Targeting CDK4/CDK6 may be an interesting therapeutic approach in CMML. The clinical value of this observation remains to be tested in forthcoming studies.
Keyword(s): Chronic myelomonocytic leukemia
Abstract: EP902
Type: E-Poster Presentation
Session title: Myelodysplastic syndromes - Biology & Translational Research
Background
In chronic myelomonocytic leukemia (CMML), therapeutic options include hematopoietic stem cell transplantation, chemotherapy, demethylating agents and hydroxyurea (HU), which is used for palliative cytoreduction. Despite these approaches, progression to secondary acute myeloid leukemia (sAML) is frequently seen and the prognosis is poor. No targeted therapies are available in CMML. CDK4 and CDK6 serve as major drug targets in applied oncology. However, the effects of CDK4/CDK6 inhibitors have not been analyzed in CMML contexts so far.
Aims
We examined the anti-neoplastic effects of HU, which has been described to counteract CDK4/CDK6-expression in leukemic cells, and the specific CDK4/CDK6 inhibitors palbociclib, ribociclib and abemaciclib on primary CMML cells and various monoblastic cell lines. We also aimed to define cooperative anti-neoplastic effects of CDK4/CDK6-targeting drugs and the multi-kinase inhibitor ponatinib.
Methods
Primary leukemic cells of the bone marrow were obtained from 11 patients: 8 with CMML, 3 with sAML following CMML. We also analyzed the monoblastic cell lines THP-1, MONO-MAC-6 (MM6) and U937. Cell proliferation was determined by 3H-thymidine uptake. Apoptosis and cell cycle distribution were quantified by flow cytometry. CDK4 and CDK6 mRNA were quantified by qPCR and protein expression and phosphorylation by Western blot analysis.
Results
Both HU and the CDK4/CDK6 inhibitors tested were found to disrupt the CDK4/CDK6-pathway in THP-1 and MM6 cells. Whereas HU decreased CDK4 and CDK6 mRNA- and protein expression, palbociclib, ribociclib and abemaciclib suppressed the expression and phosphorylation of RB1, the main target of CDK4/CDK6. HU was found to inhibit proliferation in all cell lines (IC50: THP-1: 25-50 µM; MM6: 100-150 µM; U937: 500-750 µM) and in all primary cell samples tested (IC50: 50-250 µM). Moreover, the CDK4/CDK6 inhibitors palbociclib, ribociclib and abemaciclib blocked cell proliferation in all cell lines (IC50: 0.5–10 µM) and all primary cell samples examined (IC50: 0.01–0.5 µM). Anti-proliferative effects were accompanied by cell cycle arrest and induction of apoptosis. We next searched for suitable combination partners for CDK4/CDK6 inhibitors and identified ponatinib, a broad-spectrum multi-kinase inhibitor. Ponatinib was found to inhibit proliferation in all monoblastic cell lines (IC50: 0.05-2.5 µM) and in primary leukemic cells (IC50: 0.01-2.5 µM). The drug combinations HU+ponatinib, palbociclib+ponatinib, ribociclib+ponatinib and abemaciclib+ponatinib resulted in strong synergistic anti-proliferative effects in THP-1, MM6 and primary cells isolated from 2 CMML patients. Interestingly, the combinations HU+palbociclib, HU+ribociclib, and HU+abemaciclib also resulted in synergistic growth inhibition, a phenomenon, that can be best explained by different drug target interaction profiles. Finally, we were able to demonstrate, that the triple combination `HU+palbociclib+ponatinib´ exerts superior growth-inhibitory effects in THP-1 cells compared to all 2-drug combinations tested.
Conclusion
HU and specific CDK4/CDK6-targeting drugs inhibit the proliferation of CMML cells and synergize with ponatinib in producing growth-arrest. Targeting CDK4/CDK6 may be an interesting therapeutic approach in CMML. The clinical value of this observation remains to be tested in forthcoming studies.
Keyword(s): Chronic myelomonocytic leukemia