Molecular Alterations in Bone Marrow Mesenchymal Stroma Cells of AML Patients

It is increasingly recognized that the tumor microenvironment plays a pivotal role in cancer initiation and progression. In mouse models it was shown that a genetically altered bone marrow (BM) micro milieu was sufficient to induce leukemia (Raaijmakers, Nature 2010); however, the pathogenic role and contribution of the BM stroma in leukemia initiation and during disease progression warrants further investigation. To address this, we have performed gene expression, methylation, RNAseq, whole exome sequencing (WES) in BM mesenchymal stroma cells (BM-MSC) and leukemic cells from AML patients (pts) to unravel underlying molecular alterations.

We collected BM hematopoietic cells (BM-HC) as well as plastic-adherent BM-MSC from aspirates from AML pts and healthy donors (HD). BM-MSC were expanded to passage 4 and defined as CD73⁺/CD105⁺/CD271^+/low/CD45^-/CD33^-. We investigated gene expression profiles (Affymetrix) of BM-MSC from newly diagnosed AML pts (n=20) and compared these to BM-MSC from HD (n=4). BM-MSC from AML pts displayed an altered expression signature with 191and 175genesbeingsignificantly 2-fold over- and under-expressed. KEGG analysis of differentially expressed genes in BM-MSC from AML pts exhibited enrichment for TGF-ß signalling, whereas downregulated genes were enriched for cytokine receptor interactions. Several of these candidates were validated in a larger set of BM-MSC samples by RT-PCR. One putative stroma-leukemia interaction molecule, lumican (LUM) was highly overexpressed in BM-MSC (n=60) from AML pts compared to HD (n=5; p value =0.019) indicating that LUM may affect the BM niche in AML.

To explore the altered expression pattern in AML BM-MSC compared to HD BM-MSC, global methylation analyses (Illumina Infinium HumanMethylation 450 bead chip arrays) were performed in 5 AML pts where we had collected BM-HC and BM-MSC at 3 sequential time points [initial diagnosis (ID), remission (CR), relapse (REL); n=30] as well as in BM-HC and BM-MSC from HDs (n=6). A significantly different methylation profile was evident comparing AML BM-HC to the corresponding AML BM-MSC samples, the latter showing a homogenous pattern during the course of disease. When AML BM-MSC were compared to a set of HD BM-MSC, we identified 2416 differentially methylated CpG sites (p value <0.01) indicating that an epigenetic deregulation contributes to the altered gene expression profile observed in AML BM-MSC.

These 30 AML BM-MSC/BM-HC samples were subsequently analyzed by WES to unravel genetic alterations in the compartments of the mesenchymal and hematopoietic cell fractions. In WES (HiSeq2000, 100bp paired-end), we obtained an average of 100 reads for the target region; more than 90% of the exome target region was covered at least 30-fold. When the AML BM-HC CR sample was used as germline control, a median of 3 SNVs were detected in AML BM-MSC samples. The only BM-MSC-specific alteration present in one AML patient at all time points (ID, CR, REL) was a mutation in the plectin gene (PLEC). This mutation in the ROD domain of this cytoskeletal linker protein is located in the hot spot for mutations described in epidermolysis bullosa. This mutation was validated by Sanger sequencing, however in a larger cohort of 50 AML ID BM-MSC, no additional PLEC mutation at the same position was found. The set of AML BM-MSC (n=15) samples further allowed us to identify lesions (SNVs, Indels) in the corresponding BM-HC (n=15). When we used the AML BM-MSC as germline control we identified in total 43 lesions in the AML BM-HC fractions, which were not found when the corresponding BM-HC CR sample was used as germline control. This unraveled pre-leukemic lesions present in the AML BM-HC at remission: importantly using this approach, lesions in ASXL1 (Y591*) and DNMT3A (R882H), and in another patient a DNMT3A (M880V) mutation were revealed.

In conclusion, the altered gene expression profile and methylation signature of AML BM-MSC provide novel insights into the pathogenic role of the leukemic BM microenvironment. Genetic alterations explored by WES revealed only very few genetic hits that will require further functional exploration. However, the low number of genetic alterations suggests that the transcriptional and epigenetic alterations are directed by extrinsic factors. At the same time, AML BM-MSC provides a non-hematopoietic derived germline control that allows to unravel pre-leukemic lesions in BM-HC.