Abstract
Abstract 790
Lenalidomide has emerged as a very effective therapy for del(5q) myelodysplasia. Its mechanism of action, however, has hitherto remained elusive. Interestingly, the more primitive hematopoietic compartment seems to possess a clonal advantage where del(5q) HSC are able to outcompete remaining normal HSC. We have previously demonstrated that lenalidomide is able to abrogate this clonal advantage and found that lenalidomide restored expression of the matricellular protein SPARC, a gene located within the commonly deleted region on chromosome 5q.
We hypothesized that the decreased expression of SPARC in del(5q) HSC leads to increased adhesion of HSC to their respective niche cells, translating to increased rates of proliferation, partly explaining the competitive advantage against non-del(5q) HSC.
We conducted a prospective study analyzing the del(5q) HSC/progenitor compartment from 23 patients before, during and after (refractory phase) lenalidomide, in order to test whether a hematopoietic stem cell (HSC)-intrinsic decrease of SPARC explains the why and how a clone of cells inherently defective at spawning functioning cellular descendants is not selected against, but rather exhibits a clonal advantage. In addition, we studied whether treatment with lenalidomide induced changes in the microenvironment these cells reside in.
We analyzed cell cycle distribution, frequency of apoptosis, and expression of adhesion markers on normal and del(5q) HSPC by multi-parameter flow cytometry. These experiments revealed a slight increase in proliferation of del(5q) versus normal HSC, as well as complex changes in the expression of adhesion markers in HSC of patients treated with lenalidomide.
We studied the functional adhesion of normal and del(5q) HSPC to defined matrix components of the microenvironment and observed that HSPC from del(5q) patients exhibited stronger adhesion than normal bone marrow cells to fibronectin and VCAM-1. Recombinant SPARC protein abrogated adhesion to VCAM-1 specifically in a subset of patients, while having no significant effect on normal HSPC.
To study whether SPARC plays a role in the clonal dominance, we used lentiviral transduction to overexpress SPARC in HSPC and found that increased expression of SPARC led to severely reduced engraftment in NSG-mice.
We also analyzed how lenalidomide impacts the microenvironmental niche. To this end, we compared the gene expression profile of mesenchymal stem cells (MSC) obtained from del(5q) patients by DNA microarray and found only 36 genes to be differentially expressed by more than 2-fold, with 15 and 21 genes up- or downregulated in del(5q) MSC, respectively. Using longitudinal bone marrow biopsies from 5 patients on and off treatment, we analyzed whether lenalidomide induced changes in the frequency of candidate niche cells such as mesenchymal stem cells (MSC), macrophages, endothelium and megakaryocytes, by using immunohistochemical markers for the aforementioned cells. Lenalidomide induced no significant changes in the number of nestin+ MSC but seemed to decrease the number of Factor-VIII+ megakaryocytes.
Taken together, these studies suggest that decreased expression of SPARC leads to increased adhesion of del(5q) HSC/progenitor cells to defined components of the microenvironment and may explain why del(5q) HSC are able to outcompete the remaining healthy HSC. Our studies implicate that lenalidomide is able to abrogate this clonal advantage partly via its increase in SPARC expression with a consecutive decrease in adhesion.
Disclosures:
No relevant conflicts of interest to declare.
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