Abstract

Objectives

The
bone marrow (BM) microenvironment is known to support survival of acute myeloid
leukemia (AML) cells through interactions with a network of stromal cells and
soluble factors. Accumulating evidence exists that stromal cells comprising the
BM niche release growth factors and cytokines, which play a pivotal role in response
to treatment and the development of drug resistance in AML patients. Earlier,
we demonstrated that stroma-released soluble factors were sufficient to induce significant
loss in sensitivity to several classes of drugs, including tyrosine kinase, BCL-2
and topoisomerase inhibitors (Karjalainen et al, Blood 2017). Here, we aimed to
understand the impact of BM-derived factors on the underlying molecular pathways
mediating the altered drug response in AML.

Methods

BM aspirates from AML patients (N=3) were collected after informed
consent following IRB approved protocols. For each sample, mononuclear cells were
isolated by density gradient separation, then equally divided and maintained
for 72h in Mononuclear Cell Medium (MCM; Promocell) or in RPMI medium
supplemented with 25% conditioned medium (CM) from the human BM stromal cell
line HS-5. Subsequently, RNA was extracted from the cultured AML cells, which
was depleted of ribosomal RNA and converted to cDNA libraries. The libraries
were sequenced using Illumina technology and gene expression analysis performed
as described previously (Kumar et al, BMC Genomics 2017). Differential gene
expression and gene set enrichment analyses were done to identify genes and
pathways differentially represented between the two culture conditions. Expression
of differentially regulated genes was validated by RT-qPCR for 12 patient
samples by normalizing the expression level to uncultured cells.

Results

RNA-sequenced
based gene expression analysis revealed 45 genes to be significantly
differentially expressed between the two culture conditions. Of these genes,
87% (39/45) were upregulated and 13% (6/45) down-regulated in AML cells
cultured in HS-5 CM (FDR < 0.1). Overall, hierarchical clustering showed
that stroma secreted factors had no effect on the clustering of the most
variable genes, suggesting that BM stroma-derived factors have a modest effect
on gene expression. Further analysis showed that the differentially expressed
genes are associated with cytokine-cytokine receptor interaction
and chemokine signaling pathways. Validation by RT-qPCR analysis confirmed significant
upregulation of PPBP and IL2RA in response to BM stroma-derived factors.

Conclusions

Based
on differential gene expression analysis, BM stroma-derived factors primarily induced
changes associated with cytokine signaling in AML. Additional studies are
needed to understand the impact of single gene and pathway changes on AML drug
response.