Autocrine TNF-α production supports CML stem and progenitor cell survival and enhances their proliferation

Abstract

Chronic myeloid leukemia (CML) stem cells are not dependent on BCR-ABL kinase for their survival, suggesting that kinase-independent mechanisms must contribute to their persistence. We observed that CML stem/progenitor cells (SPCs) produce tumor necrosis factor-α (TNF-α) in a kinase-independent fashion and at higher levels relative to their normal counterparts. We therefore investigated the role of TNF-α and found that it supports survival of CML SPCs by promoting nuclear factor κB/p65 pathway activity and expression of the interleukin 3 and granulocyte/macrophage-colony stimulating factor common β-chain receptor. Furthermore, we demonstrate that in CML SPCs, inhibition of autocrine TNF-α signaling via a small-molecule TNF-α inhibitor induces apoptosis. Moreover TNF-α inhibition combined with nilotinib induces significantly more apoptosis relative to either treatment alone and a reduction in the absolute number of primitive quiescent CML stem cells. These results highlight a novel survival mechanism of CML SPCs and suggest a new putative therapeutic target for their eradication.

Figure 1

Autocrine TNF-α production in CML SPCs is BCR-ABL kinase-independent, induces NFκB/p65 activity, and promotes their survival. (A) TNF-α blood plasma levels were measured by enzyme-linked immunosorbent assay in CP (n = 24) and accelerated phase (AP) (n = 3) CML patients. Levels are expressed as picograms per milliliter. The range of TNF-α blood plasma levels in normal controls (n = 8) is shown in the shaded area. (B) TNF-α mRNA expression levels were measured by qRT-PCR and normalized to the control genes ATP5B, B2M, ENOX2, GUSB, TBP, and TYW1 mRNA expression levels in newly diagnosed CP CML (n = 30) and normal (n = 4) CD34⁺ cells. (C) TNF-α protein expression was measured by intracellular flow cytometry in CML (n = 6) and normal (n = 4) CD34⁺ cells and expressed as a ratio of the mean fluorescence intensity (MFI) of TNF-α antibody-stained cells over the MFI of cells stained with a matched isotype control. (D) CML CD34⁺ cells (n = 4) were either left untreated (UT) or treated with NL (5 µM) for 48 hours, and TNF-α protein expression was measured by intracellular flow cytometry, as explained in panel C. TNF-α expression levels in the NL-treated cells were expressed as a percentage of UT cells. (E) CML CD34⁺ cells (n = 3) were either left UT or treated with TNF-α inhibitor (TNF-α inh) (3 µM) or TNF-α inh (3 μM) + TNF-α (1 ng/mL). Levels of p-NFκB/p65^Ser536 were measured by intracellular flow cytometry at 24 hours, as described in panel C, and expressed as a percentage of UT. (F) IAP2 gene expression levels were measured at 24 hours by qRT-PCR after treatment, as in panel E. Differences in gene expression levels after treatment were calculated using the 2^−ΔΔCt method, after normalization within each sample of candidate gene expression levels against GAPDH and TBP expression levels. Relative quantification (RQ) of IAP2 mRNA expression after TNF-α inh treatment was then plotted as log₂ of the 2^−ΔΔCt values (with the UT cells having a value of 0 in the graph being the calibrator). (G) CML CD34⁺ cells (n = 5) were either left UT or treated with TNF-α inh (3 µM) or TNF-α inh (3 μM) + TNF-α (1 ng/mL) for 72 hours. Percentage of apoptotic cells was measured by annexin staining. (H) CML CD34⁺ cells (n = 3) were CFSE stained and then cultured as in panel G for 72 hours. Percentage of apoptotic cells within the undivided (CFSE^max) population was measured by gating on the population double-positive for maximal CFSE expression and annexin staining. All data from independent experiments are presented as mean ± standard error of the mean. *P < .05; †P < .01; ‡P < .001; ns, not significant.

Figure 2

Effects of autocrine TNF-α inhibition in combination with NL on CML SPCs survival and proliferation. (A) CML CD34⁺ cells (n = 3) were either left UT or treated with TNF-α inh (3 µM), NL (5 μM), or their combination for 72 hours before drug washout and plating in methylcellulose progenitor assays. CFC frequency based on their morphology (erythroid-burst-forming unit [BFU-E] and erythroid-colony-forming unit [CFU-E] vs granulocyte/macrophage-colony forming unit [CFU-GM]) was recorded after 12 days of culture. (B) CML CD34⁺ cells (n = 5) were cultured as in panel A for 72 hours, and the percentage of apoptotic cells was measured by annexin staining. (C) CML CD34⁺ cells (n = 4) were CFSE stained and then cultured as in panel A for 72 hours. Percentage of apoptotic cells within the undivided (CFSE^max) population was measured as explained in Figure 1H. (D) Sorted CML, BCR-ABL⁺ (by fluorescence in situ hybridization), CD34⁺ CD38⁻ cells (n = 2) were cultured as in panel A for 72 hours. Percentage of apoptotic cells was measured by annexin staining. (E) Representative flow cytometry plot of CFSE and annexin double-staining showing levels of apoptosis within the CFSE^max population in each treatment group. (F) CML CD34⁺ cells (n = 4) were treated for 72 hours, as in panel A, and the percentage of starting CD34⁺ cells recovered within each division in each treatment group was calculated by recording the number of viable cells seeded initially in each culture and their number after different treatment conditions, using levels of CFSE fluorescence to measure the percentage of cells within each division, as explained elsewhere. All data from independent experiments are presented as mean ± standard error of the mean. *P < .05; †P < .01; ‡P < .001.