Bone Marrow Microenvironment As a Potential Key Regulator of Hematopoietic Cells Stemness in Myelodysplastic Syndromes (MDS)

Background and Rationale. Nuclear Phospholipase C (PLC) beta1 plays a pivotal role in Myelodysplastic Syndromes (MDS) and Azacytidine response [Cocco, L., et al. J Lipid Res. 2015; 56: 1853-60]. Hematopoietic Stem Cells (HSCs) primarily reside within the bone marrow (BM) microenvironment, where mesenchymal stromal cells (MSCs) regulate hematopoiesis through direct interactions with HSCs or cytokine secretion [Blau O, et al. Blood. 2011; 118:5583-92]. The aim of this study was to better elucidate the role of the BM microenvironment in interacting with MDS/AML cells, with particular emphasis on investigating the correlation between PLCbeta1 modulation and the distinctive BM microenvironment response after co-culture, in terms of PLCs gene expression, myeloid differentiation markers and cytokine secretion.

Patients and Methods. As it is a very preliminary study, here we analyzed mononuclear cells obtained from 4 higher-risk MDS (RAEB-1, R-IPSS High or Very High, Complex Karyotype), who gave informed consent in accordance with the Declaration of Helsinki. Two patients were treated with Azacytidine (AZA, 75 mg/mq/die for 7 days every 28 days) and one of them reached the fifth cycle with a stable disease. All samples were obtained from the IRCCS-Institute of Hematology “L. e A. Seràgnoli”, Bologna, Italy, where clinical evaluations were also conducted. Co-cultures were established with HS-5 MSCs, while Real-Time PCR results were normalized using molecular data obtained from 10 healthy donors (HD).

PLCbeta1 overexpression/silencing in vitro model was represented by THP-1 monocytic cells subjected to lentiviral transfection. Both wild-type and PLCbeta1 overexpressing/silencing cells (THP-1 OV/KD) were co-cultured with HS-5 cells for 96 hours.

Results. Patients showed distinct basal expression patterns of PLCbeta1: higher in 1/4 MDS and lower in 3/4 MDS, as compared to HD. Irrespective of PLCbeta1 basal expression levels, or response to AZA, in 3/4 patients CD11b increased and CD14 decreased after co-culture. Only one patient, characterized by being a therapy-related MDS, displayed a reduced expression in all myeloid differentiation markers during co-culture. During AZA therapy and after co-culture, one patient showing a stable disease had a significant PLCbeta1 reduction, CD11b increase and CD14 decrease.

In THP-1 OV cells co-cultured with HS-5 cells, we observed CD11b reduction, while no significant differences in both PLCs and other myeloid differentiation markers. In contrast, THP-1 KD cells, following co-culture, exhibited a notable increase in CD11b and a decreased CD14 expression.

Moreover, THP-1 OV cells alone displayed inhibition of IL-8 secretion, that disappeared after co-culture, in favour of IL-1beta secretion. THP-1 KD cells exhibited suppressed secretion of IL-1alpha, IL-1beta, and IL-8. However, during co-culture, the production of both IL-1alpha and IL-1beta was restored.

Conclusions. Our results, that must be confirmed in a larger group of patients, show that the impact of co-culture is not associated with the baseline levels of PLCbeta1, but co-culture recapitulates the PLCbeta1 KD in vitro model and the lack of response to AZA, showing an increase of CD11b and a CD14 reduction. Moreover, the presence of MSCs reversed cytokine secretion, as in co-cultured THP1 KD cells IL-1alpha and IL-1beta were highly secreted again. Considering their multiple roles in both physiological and pathological conditions, ongoing analyses are now further investigating their role in BM microenvironment, also using MDS-MSCs. In addition, we are now exploring the involvement of BM microenvironment in maintaining leukemic cells in a more undifferentiated state and the potential implications of these mechanisms on AZA response/resistance.