The present report describes work examining the manner in which nonmalignant bone marrow stromal cells prevent acute lymphoblastic leukemia (ALL) cell death. The initial focus was on the role of stromal cell-derived C-X-C motif chemokine 12 (CXCL12). Interference with CXCL12 production by stroma or blockade of its interactions with ALL by plerixafor did increase ALL cell death and in sensitive ALLs there was synergistic effect with conventional chemotherapy drugs. However, in contrast to most reports, there was considerable heterogeneity regarding the effect between 7 unique primary ALLs, with several exhibiting no sensitivity to CXCL12 blockade. The diversity in effect was not explained by differences in the expression of ALL cell surface receptors for CXCL12. The modest and variable effects of interference with CXCL12 on ALL led to the assessment of gene expression profiles of stromal cells and ALL cells. Gene set enrichment analysis identified pathways associated with metabolism and redox reactions as potentially important in the stromal cell: leukemia cell interaction. Exploratory imaging studies demonstrated bidirectional transfer of intracellular calcien-labelled molecules and also bidirectional transfer of mitochondria between stromal cells and ALL cells, providing potential means of metabolic interdependence of stromal cells and ALL cells.
Nonmalignant marrow stroma provides a favorable microenvironment for leukemia cell survival. The mechanisms of support are not well understood. Some studies have identified roles for some stromal derived molecules including some growth factors (EGF, FGF, PDGF), adhesion molecules (VCAM, selectins, osteopontins, integrins, fibronectin), cytokines (IL3, IL6, IL7) and others (KITLG, ANGPT1, CXCL12, LGALS3). It is clearly a multifactorial mechanism. We have developed a simple but powerful experimental system of human marrow derived stromal cells co-cultured with primary human high risk ALL cells that allows us to study the molecular mechanisms. Efficient delivery of the antiapoptotic signals requires leukemia cell contact with living, metabolically active stromal cells. Figure 1 shows several conditions in which stromal cell support for ALL is abrogated: (a) when ALL contact with stroma is prevented by a transwell membrane that allows free flow of extracellular medium; (b) after formalin fixation of stromal cells and their matrix; (c) when live stromal cell protein synthesis is prevented by pretreatment of stromal cells with G418 at nonlethal concentrations; (d) when live stromal cell RNA transcription is impaired by pretreatment with triptolide. Efficient delivery of antiapoptotic signals to leukemia cells is associated with differential of genes related to the membrane and extracellular region and to regulation of cell death. We performed we performed RNASeq studies of gene expression in stromal cells and ALL cells. We made comparisons (a) between supporting cells that could (n=17) and could not (n=7) provide antiapoptotic support to ALL, and (b) between ALL cells that were (n=3) and were not (n=3) supported by stromal cells. We identified 409 genes differentially expressed in stromal cells that prevent ALL apoptosis including genes known to play roles in cell surface interactions, cell matrix interactions, growth factor binding and regulation of cell death (Table 1). We identified 458 genes upregulated in ALL cells following contact with stromal cells including genes related to interactions with the extracellular environment, purine metabolism and regulation of apoptosis (Table 2). We identified 494 genes upregulated in stromal cells following contact with ALL related to extracellular interactions, regulation of oxidation, regulation of daunorubicin, regulation of NADP and ethanol metabolism, interferon signaling and regulation of cell proliferation (Table 3). Based on these discoveries we hypothesized that important mechanisms of stromal cell support of ALL might involve direct intercellular exchange of small molecules related to energy metabolism, nucleic metabolism, and messengers regulating cell proliferation and cell apoptosis. Possible mechanisms could include gap junctions, nanotubules or exosomes. The predominant gap junction in human cells is connexin 43 (GJA1). We found that GJA1 is constitutively expressed at high levels by stromal cells, and that GJA1 expression is increased ten-fold in ALL cells upon contact with stromal cells. Rapid bidirectional exchange of intracellular molecules occurs between stromal cells and ALL. We loaded stromal cells with fluorescent calcien AM which once inside a cell cannot diffuse through the membrane but can pass through gap junctions. We cocultured ALL cells with labelled stromal for 16 hr. We observed calcien in ALL cells in physical contact with stroma, but not in ALL cells separated from labelled stroma by a transwell. When leukemia cells were labeled with calcien we saw transfer of calcien to stroma in contact with ALL but not when separated by a transwell. Interference with gap junction function increases ALL cell death. GAP27 is an 11-amino acid peptide that specifically interferes with GJA1/connexin 43 gap junctions. Figure 2 shows substantial reduction of ALL survival in cocultures containing 1 mM GAP27 peptide. We observed similar results with use of 75 micromolar carbenoxolone, an FDA approved drug that impairs gap junction function. Disclosures No relevant conflicts of interest to declare.
Background: Bone marrow stroma provides a favorable microenvironmental niche for ALL cell survival. We and others have demonstrated that bone marrow stromal cells contribute to prevention of apoptosis in ALL cells. Objective: Identify potentially "drug-able" molecules derived from marrow stromal cells that contribute to prevention of ALL cell apoptosis. Methods: We have developed an in vitro system to identify stromal gene products that deliver antiapoptotic signals to ALL cells. Primary human ALL cells are co-cultured with human bone marrow stromal cells. We manipulate stromal cells with siRNA directed against candidate stromal cell genes. Two days later the siRNA is washed out of culture and primary ALL cells are added to the stromal cells. Controls include irrelevant siRNA. Five days later we measure viability and apoptosis in ALL cells by flow cytometry. Results: (1) Knockdown of stroma cell CXCL12 or TGFBI reduces ALL survival. We performed global gene expression analysis upon human marrow stromal cells using RNASeq technology. Using bioinformatic approaches we are selecting some of the expressed stromal genes as candidates for the molecular mechanisms by which stromal cells prevent ALL apoptosis. We present preliminary results for two of our early candidates. (A) CXCL12 is a paracrine chemokine known to have activity in the marrow microenvironment upon hematopoietic cells and we hypothesized it may participate in the effect. Knockdown of CXCL12 with siRNA increased ALL cell death in the co-culture system. As measured by quantitative reverse transcriptase PCR stromal cell CXCL12 mRNA was reduced approximately 75% by siRNA treatment. Figure 1 displays representative results of the impact of CXCL12 knockdown in stromal cell on the survival of ALL cells in the coculture. The magnitude of effect was ~40% increase in ALL cell death. (B) TGFBI (transforming growth factor beta induced) is expressed by stromal cells. The gene is involved in cell-collagen interactions and we hypothesized it played a role. siRNA reduced stromal gene expression by about 90%. Figure 2 displays representative results in which ALL cell death increased by about 50%. (2) Validation of results using inhibitors to CXCL12. The gene knockdown experiments suggested a potential role for CXCL12 in prevention of ALL cell apoptosis. To further test this we tested the effect of plerixafor, a specific inhibitor of CXCL12/CXCR4 interactions, on survival of ALL. ALL cells express CXCR4. In a dose dependent manner (25 - 400 micromolar) we observed a 31-39% reduction in ALL survival in stromal co-cultures including plerixafor. Figure 3 depicts representative results with plerixafor 200 micromolar. We are evaluating small molecules to block TGFBI. (3) Potential augmentation of chemotherapy drug effects on ALL. We hypothesize that interference with stromal cell molecules that prevent apoptosis in ALL cells may increase the effectiveness of conventional antileukemia drugs. In our stromal cell/ALL coculture system we have identified the effective in vitro concentrations of the most commonly used ALL drugs. We measured the impact of combination of low dose plerixafor (LD10) and these individual drugs (used at approximately the LD50 concentrations). Figure 4 demonstrates increased antileukemia effects related to plerixafor for dexamethasone, vincristine, and 6-mercaptopurine. Results are plotted as a percentage of ALL cells surviving in the absence of any drugs. The low dose plerixafor alone control did not produce a statistically significant reduction in ALL survival. Conclusions: Marrow stromal cell-produced CXCL12 may contribute to prevention of apoptosis in human ALL cells. Pharmacological interference with its effect may enhance the effectiveness of some conventional chemotherapy drugs. Marrow stromal cell-produced TGFBI may also contribute to prevention of apoptosis in human ALL cells. Disclosures No relevant conflicts of interest to declare.
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