IFNγ directly counteracts imatinib-induced apoptosis of primary human CD34+ CML stem/progenitor cells potentially through the upregulation of multiple key survival factors

Abstract

Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon γ (IFNγ) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFNγ antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFNγ also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFNγ was independent of alternative IFNγ signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFNγ upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFNγ, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.

Keywords: Chronic myeloid leukemia; apoptosis; interferon gamma; leukemic stem cell.

Figure 1.

IFNγ neutralization markedly counteracts the anti-apoptotic effect of activated T cell-conditioned medium on imatinib-treated JURL-MK1 cells. (a) Proportion of active caspase-3 positive JURL-MK1 cells left untreated or treated with 1 µM imatinib for 22 hours in a mixture of 20% fresh non-conditioned complete medium and 80% non-activated (non-Act) or activated (Act) TCM (produced in complete medium by T cells enriched from the peripheral blood of two healthy donors; D1, donor No. 1; D2, donor No. 2), in the absence (-) or presence (+) of isotype control (ctrl) or neutralizing anti-human IFNy antibodies (Ab) to a final concentration of 10 μg/ml each, and analyzed by flow cytometry. (b) Proportion of active caspase-3/7 positive JURL-MK1 cells left untreated (-) or treated (+) with 1 µM imatinib for 36 hours in a mixture of 95% fresh non-conditioned complete medium and 5% non-activated (non-Act) or activated (Act) TCM (produced in StemSpan SFEM medium by T cells enriched from the peripheral blood of a third healthy donor; D3, donor No. 3), in the absence (-) or presence (+) of isotype control (ctrl) and/or neutralizing anti-human IFNy and/or anti-human GM-CSF antibodies (Ab) to a final concentration of 20 μg/ml each, and analyzed by fluorescence live cell microscopy. The IFNy concentration in the conditioned mediums produced by the non-activated and activated T cells of donor No. 3 were undetectable and 95,2 ng/ml, respectively. Imatinib-induced apoptosis was faster in the batch (No. 1) of JURL-MK1 shown in panel A than in the batch (No. 2) shown in panel B. Data represent mean with standard deviation derived from three (a) or two (b) technical replicates.

Figure 2.

IFNγ exerts a rapid anti-apoptotic effect on imatinib-treated CML cells. Proportion of active caspase-3/7 positive JURL-MK1 (batch No. 1) (a) and K562 (b) cells left untreated or treated for the hours indicated with 1 μM imatinib and/or 5 ng/ml IFNγ, analyzed by fluorescence live cell microscopy. Black line, untreated; green line, IFNγ; red line, imatinib; blue line, imatinib and IFNγ. Data represent mean with standard deviation derived from three technical replicates. Experiments were performed at least twice with similar results.

Figure 3.

Reducing STAT1 protein level markedly inhibits the anti-apoptotic effect of IFNγ on imatinib-treated JURL-MK1 cells. (a) Proportion of active caspase-3/7 positive JURL-MK1 (batch No. 1) cells left untreated (-) or treated (+) for 18 hours with 1 μM imatinib and/or 5 ng/ml IFNγ in the absence (-) or presence (+) of 100 nM wortmannin or 10 μM SC75741 or 500 nM ralimetinib, analyzed by fluorescence live cell microscopy. (b) Immunoblot analysis of STAT1 and β-actin protein expression in total cell extracts of JURL-MK1 (batch No. 1) cells transfected with a negative control or a STAT1-specific siRNA. (c) Proportion of active caspase-3 positive JURL-MK1 (batch No. 1) cells transfected with a negative control or a STAT1-specific siRNA, and then left untreated (-) or treated (+) for 18 hours with 1 μM imatinib and/or 5 ng/ml IFNγ. Data represent mean with standard deviation derived from three (a) or four (c) independent experiments. Representative blots are shown. ns, not significant; *P < .05; **P < .01.

Figure 4.

Activated T cell-conditioned medium increases proliferation and counteracts imatinib-induced apoptosis of primary human CD34+ CML stem/progenitor cells. Relative numbers of viable (a), and proportion of active caspase-3/7 positive (b) primary human CD34+ CML stem/progenitor cells (obtained from the peripheral blood of Patients Nos. 1, 2 (a) and 3 (b)), left untreated or treated for the hours indicated with 5 μM imatinib in a mixture of 80% fresh non-conditioned StemSpan SFEM medium and 20% non-activated (non-Act) or activated (Act) TCM (the same TCMs (D3) used in the experiment shown in Figure 1b), and analyzed by cell counting (a) or fluorescence live cell microscopy (b). Black line, non-activated TCM; green line, activated TCM; blue line, non-activated TCM and imatinib; orange line, activated TCM and imatinib. Data represent mean with standard deviation derived from three technical replicates.

Figure 5.

The anti-apoptotic effect of activated T cell secretome on primary human CD34+ CML stem/progenitor cells is strongly counteracted by IFNγ neutralization during the first 36 hours, while at later time-points simultaneous IFNγ and GM-CSF neutralization partially blocks the anti-apoptotic effect. Proportion of active caspase-3/7 positive primary human CD34+ CML stem/progenitor cells (obtained from the peripheral blood of Patient No. 2) left untreated or treated for the indicated hours with 5 µM imatinib in a mixture of 95% fresh non-conditioned StemSpan SFEM medium and 5% non-activated (non-Act) or activated (Act) TCM (the same TCMs (D3) used in the experiments shown in Figure 1b and Figure 4), in the presence of only the two isotype controls (solid lines), or neutralizing anti-human IFNy and rat IgG2a isotype control (dotted lines), or neutralizing anti-human GM-CSF and mouse IgG1 isotype control (dashed lines), or the combination (dash-dotted lines) of the two neutralizing antibodies (Ab) to a final concentration of 20 μg/ml each, and analyzed by fluorescence live cell microscopy. Black line, non-activated TCM; green line, activated TCM; blue line, non-activated TCM and imatinib; orange line, activated TCM and imatinib. Data represent mean with standard deviation derived from two technical replicates.

Figure 6.

The MCL1 inhibitor A1210477 only partially blocks the anti-apoptotic effect of IFNγ on imatinib-treated primary human CD34+ CML stem/progenitor cells (a) Immunoblot analysis of MCL-1L and β-actin protein expression in total cell extracts of primary human CD34+ CML stem/progenitor cells (obtained from the peripheral blood of Patient No. 4) left untreated (-) or treated (+) for 18 hours with 5 μM imatinib and/or 5 ng/ml IFNγ. (b) Proportion of activated caspase-3 positive primary human CD34+ CML stem/progenitor cells (obtained from the peripheral blood of Patient No. 4) left untreated (-) or treated (+) for 24 hours with 5 μM imatinib and/or 5 ng/ml IFNγ in the absence or presence of 10 μM A1210477. Data represent mean with standard deviation derived from four technical replicates.

Figure 7.

IFNγ upregulates the expression of several key anti-apoptotic genes in imatinib-treated primary human CD34+ CML stem/progenitor cells. (a) Relative mRNA levels of selected key anti-apoptotic genes normalized to EEF1A1, quantified by real-time RT-PCR in primary human CD34+ CML stem/progenitor cells (obtained from the peripheral blood of Patient No. 4) left untreated or treated for 18 hours with 5 μM imatinib (IM) and/or 5 ng/ml IFNγ. (b) Relative mRNA levels of selected key anti-apoptotic genes normalized to EEF1A1, quantified by real-time RT-PCR in primary human CD34+ CML stem/progenitor cells (obtained from the peripheral blood of Patient No. 5) left untreated or treated for 90 minutes with 5 μM imatinib (IM) and/or 5 ng/ml IFNγ in the absence (w/o cytokines) or presence of SCF, FL, and TPO. (c) Relative mRNA levels of selected key anti-apoptotic genes normalized to EEF1A1, quantified by real-time RT-PCR in primary human CD34+ CML stem/progenitor cells (obtained from the peripheral blood of Patient No. 6) left untreated or treated for 90 minutes with 5 μM imatinib (IM) and/or 5 ng/ml IFNγ in the absence of cytokines. (d) Immunoblot analysis of BCL-XL, MCL-1L, PARP9, PARP14 and β-actin protein expression in total cell extracts of CD34+ stem/progenitor cells (obtained from the peripheral blood of Patients Nos. 2, 3 and 7) left untreated (-) or treated (+) for 18 hours with 5 μM imatinib and/or 5 ng/ml IFNγ. PIM1 and PIM2 proteins were expressed in extremely low amounts, or were not detectable in any of the lysates (not shown).