Contributions
Abstract: EP904
Type: E-Poster Presentation
Session title: Myelodysplastic syndromes - Biology & Translational Research
Background
Myelodysplastic Syndrome (MDS) is characterized by ineffective clonal hematopoiesis and bone marrow failure (BMF). Azacitidine (AZA), a hypomethylating agent is the standard of care for treatment of higher-risk MDS patients either alone or in combination with newer therapeutic options [Silverman LR, Cancer Medicine 2017]. Responses to AZA alone occur in 50% of pts with significant improvement in hematopoiesis [Fenaux et al Lancet Oncol 2009]. AZA improves overall survival of pts, yet despite this, 100% of pts ultimately fail treatment. AZA combined with Rigosertib (RIGO), a novel agent that downregulates the Ras pathway inhibits Ras/Raf signaling [Athuluri-Divakar SK, et al. Cell, 2016], yields a response rate of 54% in pts who were HMA failures [Navada SC, et al. EHA 2019]. Chronic activation of the MAPK pathway can activate inflammatory pathways suppressing hematopoietic function (Ramalingham Nat Comm 2020). Thus, understanding the mechanism of resistance and identification of targets which can reverse HMA failure and improve hematopoiesis in MDS pts is critical.
Aims
To assess the effects of modulation of the Ras pathway combined with a hypomethylating agent specifically on MAPK signaling, the stemness and differentiation of the MDS-L cell line.
Methods
We investigated the differential protein expression in response to different treatment (AZA, RIGO alone and sequential combinations RIGO/AZA; AZA/RIGO) in vitro in MDS-L cell line by Reverse Phase Protein Array (RPPA). Further, we also studied the functional role of these treatments on differentiation in MDS cells by growing cells on semi-solid media as well as by flow cytometry using various stem cell and differentiation markers (CD34, CD38, CD45, CD123).
Results
RPPA analysis indicated discrete responses to treatment in MDS-L cells. The response was prominent with differential expression of 43 proteins specifically in MDS-L cells treated with the sequenced combination of RIGO/AZA that include PIK3R1, AKT1, mTOR, p38 MAPK, PTEN, RPS6KA1 (Fold change (FC) < -2). All of these proteins are downregulated which suggests inhibition of PI3K, mTOR and MAPK signaling. Proteins belonging to metabolic pathways, including ACC1 and ACLY, were found to be downregulated, whereas proteins related to mitochondrial function and oxidative phosphorylation (OXPHOS) were upregulated (FC > 1.5/< -1.5) in cells treated with RIGO/AZA.In addition, we found that MDS-L cells represented both CD34+CD38+ and CD34+CD38- populations. AZA increases the percentage of CD34+CD38+, indicative of differentiation, whereas RIGO alone increased the percentage of CD34+CD38- cells, representing a primitive stem cell population. Based on the combination of differentiation markers, we observed that RIGO alone, and sequenced combination RIGO/AZA, impacts different progenitors such as granulocyte-macrophage progenitor (GMP), megakaryocyte erythroid progenitor (MEP), and multipotent progenitor (MPP). RIGO/AZA treatment showed a decrease in GMP, while the % of MEP was increased, as compared to other treatments.
Conclusion
In addition to inhibition of the PI3/AKT/mTOR and MAPK pathway, the RIGO/AZA combination impacts metabolic and differentiation pathways of MDS-L cells. RIGO alone appears to promote maintenance of a primitive stem cell population in a less differentiated state, while the RIGO/AZA sequenced combination appears to encourage cells to enter a cycling stage. Further studies are underway to determine the effect of metabolic changes on differentiation and maintenance of hematopoietic stem cells.
Keyword(s): Azacitidine, MAP kinase, Myelodysplasia, Ras
Abstract: EP904
Type: E-Poster Presentation
Session title: Myelodysplastic syndromes - Biology & Translational Research
Background
Myelodysplastic Syndrome (MDS) is characterized by ineffective clonal hematopoiesis and bone marrow failure (BMF). Azacitidine (AZA), a hypomethylating agent is the standard of care for treatment of higher-risk MDS patients either alone or in combination with newer therapeutic options [Silverman LR, Cancer Medicine 2017]. Responses to AZA alone occur in 50% of pts with significant improvement in hematopoiesis [Fenaux et al Lancet Oncol 2009]. AZA improves overall survival of pts, yet despite this, 100% of pts ultimately fail treatment. AZA combined with Rigosertib (RIGO), a novel agent that downregulates the Ras pathway inhibits Ras/Raf signaling [Athuluri-Divakar SK, et al. Cell, 2016], yields a response rate of 54% in pts who were HMA failures [Navada SC, et al. EHA 2019]. Chronic activation of the MAPK pathway can activate inflammatory pathways suppressing hematopoietic function (Ramalingham Nat Comm 2020). Thus, understanding the mechanism of resistance and identification of targets which can reverse HMA failure and improve hematopoiesis in MDS pts is critical.
Aims
To assess the effects of modulation of the Ras pathway combined with a hypomethylating agent specifically on MAPK signaling, the stemness and differentiation of the MDS-L cell line.
Methods
We investigated the differential protein expression in response to different treatment (AZA, RIGO alone and sequential combinations RIGO/AZA; AZA/RIGO) in vitro in MDS-L cell line by Reverse Phase Protein Array (RPPA). Further, we also studied the functional role of these treatments on differentiation in MDS cells by growing cells on semi-solid media as well as by flow cytometry using various stem cell and differentiation markers (CD34, CD38, CD45, CD123).
Results
RPPA analysis indicated discrete responses to treatment in MDS-L cells. The response was prominent with differential expression of 43 proteins specifically in MDS-L cells treated with the sequenced combination of RIGO/AZA that include PIK3R1, AKT1, mTOR, p38 MAPK, PTEN, RPS6KA1 (Fold change (FC) < -2). All of these proteins are downregulated which suggests inhibition of PI3K, mTOR and MAPK signaling. Proteins belonging to metabolic pathways, including ACC1 and ACLY, were found to be downregulated, whereas proteins related to mitochondrial function and oxidative phosphorylation (OXPHOS) were upregulated (FC > 1.5/< -1.5) in cells treated with RIGO/AZA.In addition, we found that MDS-L cells represented both CD34+CD38+ and CD34+CD38- populations. AZA increases the percentage of CD34+CD38+, indicative of differentiation, whereas RIGO alone increased the percentage of CD34+CD38- cells, representing a primitive stem cell population. Based on the combination of differentiation markers, we observed that RIGO alone, and sequenced combination RIGO/AZA, impacts different progenitors such as granulocyte-macrophage progenitor (GMP), megakaryocyte erythroid progenitor (MEP), and multipotent progenitor (MPP). RIGO/AZA treatment showed a decrease in GMP, while the % of MEP was increased, as compared to other treatments.
Conclusion
In addition to inhibition of the PI3/AKT/mTOR and MAPK pathway, the RIGO/AZA combination impacts metabolic and differentiation pathways of MDS-L cells. RIGO alone appears to promote maintenance of a primitive stem cell population in a less differentiated state, while the RIGO/AZA sequenced combination appears to encourage cells to enter a cycling stage. Further studies are underway to determine the effect of metabolic changes on differentiation and maintenance of hematopoietic stem cells.
Keyword(s): Azacitidine, MAP kinase, Myelodysplasia, Ras