Contributions
Abstract: EP426
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Receptor tyrosine kinases FLT3 and KIT are mutated in up to 40% of AML patients. Mutant receptor tyrosine kinases (RTK) are promising targets for cancer treatment. Especially, selective FLT3 inhibitors (quizartinib, sorafenib, midostaurin, etc) show significant clinical activity in AML patients. However, relapses associated with the acquired drug resistance by leukemia cells remain major limitation of the monotherapy with RTK inhibitors.
Aims
We aimed at identifying cytokines and growth factors mediating response of leukemia cells to RTK inhibitors to better understand the mechanisms of therapy escape by leukemia cells.
Methods
We used a panel of human leukemia cells lines - Kasumi-1, MV4;11, HL-60, Kg1a, THP-1 and stroma-derived cell line - HS-5. qPCR was used to measure cytokine and growth-factor receptors expression at the mRNA level in cells treated with inhibitors. Confocal microscopy and flow cytometry were applied to detect KIT, FLT3, TrkA and IL3-R proteins at the protein level. By the lentiviral transduction we obtained cells with the overexpression of mutant KIT (N822K) and FLT3-iTD based on the murine cytokine-dependent cell line FDC-P1. We also established ERK-KTR reporter cell lines based on the HS-5 and THP-1 cells to study MAPK activity in living stroma and macrophages at the single-cell level when co-cultivated with leukemia cells. THP-1 cells were differentiated into macrophages with phorbol 12-myristate 13-acetate.
Results
Up-regulation of IL-3R and NTRK1 (encoding TrkA) in response to RTK targeted therapy was a unique feature of AML cells with mutant RTK - FLT3 and KIT. Recombinant IL-3 rescued Kasumi-1 and MV4;11 cells from KIT and FLT3 inhibition, respectively, and this effect was enhanced by NGF (ligand of TrkA). Overexpression of mutant KIT in IL-3-dependent FDC-P1 resulted in the up-regulation of IL-3R, that points to the direct link between mutant RTKs and IL-3R at the transcription level. Combination of RTK inhibitors with SRC (bosutnib and bafetinib) and JAK2 (ruxolitinib), but not MAPK targeting drugs, abrogated IL-3 rescue-effect on AML cells. Moreover, SRC and JAK2 inhibition sensitized Kasumi-1 and MV4;11 cells to several RTK inhibitors. Notably, the toxic effect of RTK inhibition with respect to leukemia cells was attenuated in the presence of HS-5 or THP-1 cells. RTK and downstream kinase inhibitors differently changed ERK activity in stroma cells and model macrophages in the presence or abscence of leukemia cells. Thus, dual targeting of RTK and SRC or JAK2 is promising for the effective suppression of leukemia cells proliferation via abrogation of cytokine-mediated death-escape by leukemia cells.
Conclusion
IL-3 and NGF play a vital role in the development of therapy-resistant AML cells with mutant RTKs. Studies with patient-derived xenotransplantation models are needed to clarify the findings. All in all, our study provides a rational for the use of RTK inhibitors in combination with agents blocking compensatory cytokine and growth factor signaling.
Keyword(s): Acute myeloid leukemia, IL-3, Receptor tyrosine kinase
Abstract: EP426
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
Receptor tyrosine kinases FLT3 and KIT are mutated in up to 40% of AML patients. Mutant receptor tyrosine kinases (RTK) are promising targets for cancer treatment. Especially, selective FLT3 inhibitors (quizartinib, sorafenib, midostaurin, etc) show significant clinical activity in AML patients. However, relapses associated with the acquired drug resistance by leukemia cells remain major limitation of the monotherapy with RTK inhibitors.
Aims
We aimed at identifying cytokines and growth factors mediating response of leukemia cells to RTK inhibitors to better understand the mechanisms of therapy escape by leukemia cells.
Methods
We used a panel of human leukemia cells lines - Kasumi-1, MV4;11, HL-60, Kg1a, THP-1 and stroma-derived cell line - HS-5. qPCR was used to measure cytokine and growth-factor receptors expression at the mRNA level in cells treated with inhibitors. Confocal microscopy and flow cytometry were applied to detect KIT, FLT3, TrkA and IL3-R proteins at the protein level. By the lentiviral transduction we obtained cells with the overexpression of mutant KIT (N822K) and FLT3-iTD based on the murine cytokine-dependent cell line FDC-P1. We also established ERK-KTR reporter cell lines based on the HS-5 and THP-1 cells to study MAPK activity in living stroma and macrophages at the single-cell level when co-cultivated with leukemia cells. THP-1 cells were differentiated into macrophages with phorbol 12-myristate 13-acetate.
Results
Up-regulation of IL-3R and NTRK1 (encoding TrkA) in response to RTK targeted therapy was a unique feature of AML cells with mutant RTK - FLT3 and KIT. Recombinant IL-3 rescued Kasumi-1 and MV4;11 cells from KIT and FLT3 inhibition, respectively, and this effect was enhanced by NGF (ligand of TrkA). Overexpression of mutant KIT in IL-3-dependent FDC-P1 resulted in the up-regulation of IL-3R, that points to the direct link between mutant RTKs and IL-3R at the transcription level. Combination of RTK inhibitors with SRC (bosutnib and bafetinib) and JAK2 (ruxolitinib), but not MAPK targeting drugs, abrogated IL-3 rescue-effect on AML cells. Moreover, SRC and JAK2 inhibition sensitized Kasumi-1 and MV4;11 cells to several RTK inhibitors. Notably, the toxic effect of RTK inhibition with respect to leukemia cells was attenuated in the presence of HS-5 or THP-1 cells. RTK and downstream kinase inhibitors differently changed ERK activity in stroma cells and model macrophages in the presence or abscence of leukemia cells. Thus, dual targeting of RTK and SRC or JAK2 is promising for the effective suppression of leukemia cells proliferation via abrogation of cytokine-mediated death-escape by leukemia cells.
Conclusion
IL-3 and NGF play a vital role in the development of therapy-resistant AML cells with mutant RTKs. Studies with patient-derived xenotransplantation models are needed to clarify the findings. All in all, our study provides a rational for the use of RTK inhibitors in combination with agents blocking compensatory cytokine and growth factor signaling.
Keyword(s): Acute myeloid leukemia, IL-3, Receptor tyrosine kinase