Contributions
Abstract: PB1633
Type: Publication Only
Session title: Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background
Multiple myeloma (MM) plasma cells (PC) growth and the associated bone disease is tightly dependent on bone marrow (BM) microenvironment. Survival pathways, such as NF-κB, Hedgehog and Wnt/β-catenin, support the MM clone and bone homeostasis imbalance. The Wnt/β-catenin pathway is pivotal for bone formation since it stimulates RUNX2 expression, the master gene regulator of osteoblastogenesis. It has been shown that MM PC cells inhibit RUNX2 activity in mesenchymal stromal cells (MSC). Moreover, RUNX2 overexpression in MM cells is also associated to osteoblastogenesis inhibition. Protein kinase CK1α supports MM PC growth and it is a known target of Lenalidomide (Lena). Our preliminary data show that CK1α gene silencing in MSC sustains RUNX2 gene expression, favoring the osteogenic program.
It is known that Lena works through a novel drug mechanism, modulating Cereblon E3 ubiquitin ligase. It induces the ubiquitination and the subsequent proteasomal degradation of Ikaros, Aiolos and CK1α leading to MM cells death. The effects of the Lena-CK1a axis on the MSC osteogenic potential and the MM BM-dependent growth are unknown.
Aims
To investigate the effects of Lena-induced CK1α degradation in MSC on: 1) cell survival. 2) the MSC osteoblastic phenotype, including RUNX2 and ALP expression.
To study the impact of Lena in a MSC-MM PC crosstalk BM microenvironment culture model on: 1) MM PC growth/survival 2) the MM PC-induced anti-osteogenic phenotype.
Methods
Cell lines and cultures: the IL-6 dependent INA-6 MM cell line and the MSC-hTERT stromal cell line have been used. A BM microenvironment model was obtained by plating INA-6 on a layer of MSC-hTERT cells. MM cells and MSC pure populations were obtained by cell sorting since MSC express GFP. Primary BM stromal cells were isolated from MM and MGUS sample patients. All the experiments were performed adding Lena to the culture medium. The rate of apoptosis was detected through Annexin V and Propidium Iodide staining. qRT-PCR and WB analysis were used to analyze mRNA and protein level expression of CK1α, RUNX2 and other osteogenic markers.
Results
Lena treatment did not change MSC viability, while it induced MM cells apoptosis. CK1α protein level was reduced after Lena treatment, not only in MM cells, but also in MSC cultured alone or with PC. Surprisingly, despite CK1α degradation, both RUNX2 and ALP mRNA expression was reduced in MSC-hTERT and preliminarly in primary MSC, suggesting a negative effect of Lena on osteogenic differentiation. In the MM-BM stromal cells co-culture model, we found reduced RUNX2 expression levels both in MM cells and in MSC
Conclusion
Lena induced CK1α reduction does not seem to be sufficient to recover the MSC osteogenic program, while it is sufficient to reduce RUNX2 expression in MM cells, inducing cell death. Further studies will be necessary to investigate if combining CK1α silencing/chemical inhibition to Lena, in the co-culture model system, could be promising not only to cause PC death, but also to rescue the MSC osteoblastic phenotype.
Keyword(s): Bone disease, Immunomodulatory thalidomide analog, Kinase, Multiple myeloma
Abstract: PB1633
Type: Publication Only
Session title: Myeloma and other monoclonal gammopathies - Biology & Translational Research
Background
Multiple myeloma (MM) plasma cells (PC) growth and the associated bone disease is tightly dependent on bone marrow (BM) microenvironment. Survival pathways, such as NF-κB, Hedgehog and Wnt/β-catenin, support the MM clone and bone homeostasis imbalance. The Wnt/β-catenin pathway is pivotal for bone formation since it stimulates RUNX2 expression, the master gene regulator of osteoblastogenesis. It has been shown that MM PC cells inhibit RUNX2 activity in mesenchymal stromal cells (MSC). Moreover, RUNX2 overexpression in MM cells is also associated to osteoblastogenesis inhibition. Protein kinase CK1α supports MM PC growth and it is a known target of Lenalidomide (Lena). Our preliminary data show that CK1α gene silencing in MSC sustains RUNX2 gene expression, favoring the osteogenic program.
It is known that Lena works through a novel drug mechanism, modulating Cereblon E3 ubiquitin ligase. It induces the ubiquitination and the subsequent proteasomal degradation of Ikaros, Aiolos and CK1α leading to MM cells death. The effects of the Lena-CK1a axis on the MSC osteogenic potential and the MM BM-dependent growth are unknown.
Aims
To investigate the effects of Lena-induced CK1α degradation in MSC on: 1) cell survival. 2) the MSC osteoblastic phenotype, including RUNX2 and ALP expression.
To study the impact of Lena in a MSC-MM PC crosstalk BM microenvironment culture model on: 1) MM PC growth/survival 2) the MM PC-induced anti-osteogenic phenotype.
Methods
Cell lines and cultures: the IL-6 dependent INA-6 MM cell line and the MSC-hTERT stromal cell line have been used. A BM microenvironment model was obtained by plating INA-6 on a layer of MSC-hTERT cells. MM cells and MSC pure populations were obtained by cell sorting since MSC express GFP. Primary BM stromal cells were isolated from MM and MGUS sample patients. All the experiments were performed adding Lena to the culture medium. The rate of apoptosis was detected through Annexin V and Propidium Iodide staining. qRT-PCR and WB analysis were used to analyze mRNA and protein level expression of CK1α, RUNX2 and other osteogenic markers.
Results
Lena treatment did not change MSC viability, while it induced MM cells apoptosis. CK1α protein level was reduced after Lena treatment, not only in MM cells, but also in MSC cultured alone or with PC. Surprisingly, despite CK1α degradation, both RUNX2 and ALP mRNA expression was reduced in MSC-hTERT and preliminarly in primary MSC, suggesting a negative effect of Lena on osteogenic differentiation. In the MM-BM stromal cells co-culture model, we found reduced RUNX2 expression levels both in MM cells and in MSC
Conclusion
Lena induced CK1α reduction does not seem to be sufficient to recover the MSC osteogenic program, while it is sufficient to reduce RUNX2 expression in MM cells, inducing cell death. Further studies will be necessary to investigate if combining CK1α silencing/chemical inhibition to Lena, in the co-culture model system, could be promising not only to cause PC death, but also to rescue the MSC osteoblastic phenotype.
Keyword(s): Bone disease, Immunomodulatory thalidomide analog, Kinase, Multiple myeloma