Contributions
Abstract: EP657
Type: E-Poster Presentation
Session title: Chronic myeloid leukemia - Biology & Translational Research
Background
Treatment with tyrosine kinase inhibitors (TKIs) substantially improved the clinical outcome in CML. Although, the Bcr-Abl1-directed approach eliminates the majority of CML cells, TKIs often spare leukemic stem cells (LSCs). Interestingly, tumor necrosis factor (TNF) has been described to coordinate hematopoietic stem cell survival and a TKI persistent TNF signature in LSCs was identified. Elevated TNF plasma levels were observed in chronic phase (CP) and progressed CML and were significantly elevated in bone marrow and spleen in CML mouse models. Targeting the TNF-induced NFᴋB-signaling by overexpressing the inhibitory IᴋBα protein, impaired leukemogenesis in a retroviral model of Bcr-Abl1 driven disease.
Aims
Here, we aimed to complement TKI therapy by TNF pathway-directed treatment, to specifically target persisting CML cells in CP- and blast crisis (BC) CML.
Methods
Publicly available microarray data were analyzed for induction of TNF regulated pathways. 32D Bcr-Abl1, SCLtTA/Bcr-Abl1-BM, human K562 & KCL22 (imatinib-sensitive (S) and -resistant (R)) cells were evaluated using qRT-PCR, Western blot, annexin V & CFSE staining. CP-CML-CD34+-cells and BC mononuclear cells (MNCs) were subjected to colony-forming unit (CFU) assays or annexin V + CFSE measurements upon treatment with the IKK2 small molecule inhibitor LY2409881 (LY) +/- nilotinib or DMSO as control, in the presence of TNF, IL1α or IL1β.
Results
Analysis of microarray data unveiled enrichment of TNF signaling molecules in the CD34+CD38- compartment of CP-CML patient cells. 32D Bcr-Abl1 and primary murine SCLtTA Bcr-Abl1-BM cells exhibited enhanced TNF signaling compared to their Bcr-Abl1 negative counterparts. 32D Bcr-Abl1, KCL22 and K562 cells displayed no reduction in TNF signaling molecules upon TKI treatment, suggesting a Bcr-Abl1 kinase-independent persistence. The combination of nilotinib and LY reduced expression of IᴋBα and NFᴋB and abrogated their phosphorylation. This treatment reduced proliferation and induced apoptosis (fold change [fc]: 2.4; p<0.01) in KCL22 S cells. In primary CD34+ CP-CML cells, we discovered diminished clonogenic potential compared to TKI alone (fc: 0.05; p<0.01). Replating revealed nilotinib to preserve cells with colony-forming ability, as previously reported. These LSCs were in turn targeted by IKK2 inhibition (fc: 0.17; p<0.01). Reduction of the clonogenic potential was accompanied by apoptosis induction upon combinatorial treatment compared to TKI alone (fc: 2.5; p<0.01), also in the non-dividing cell fraction (fc: 5.2; p<0.001). Gene set enrichment of microarray data comparing BC to CP patient cells displayed further upregulation of TNF signaling in BC-LSCs. Interestingly, IKK2 inhibition could bypass TKI resistance in KCL22 R cells by apoptosis induction (fc: 2.4; p<0.01). Also, in BC-CML MNCs, combined nilotinib and LY treatment significantly decreased self-renewal (fc: 0.1; p<0.01) and induced apoptosis (fc: 1.8; p<0.01). Furthermore, combinatorial treatment targeted persistent CML-CP LSCs in the presence of IL1α and IL1β (fc: 0.6 & 0.5; p<0.05), underlining the relevance of IKK2 inhibition as global approach to target the pro-leukemic inflammatory environment.
Conclusion
In conclusion, we demonstrate that elevated TNF signaling in CML patients persists during TKI treatment. By combining IKK2 inhibition with a Bcr-Abl1-directed TKI, we were able to target non-dividing CP-CML LSCs but also TKI resistant BC-CML cells. We suggest targeting of TNF-induced signaling as a promising approach to address TKI persistence and resistance in CP- and BC-CML.
Keyword(s): Blast crisis, Chronic myeloid leukemia, Drug resistance, Tumor necrosis factor (TNF)
Abstract: EP657
Type: E-Poster Presentation
Session title: Chronic myeloid leukemia - Biology & Translational Research
Background
Treatment with tyrosine kinase inhibitors (TKIs) substantially improved the clinical outcome in CML. Although, the Bcr-Abl1-directed approach eliminates the majority of CML cells, TKIs often spare leukemic stem cells (LSCs). Interestingly, tumor necrosis factor (TNF) has been described to coordinate hematopoietic stem cell survival and a TKI persistent TNF signature in LSCs was identified. Elevated TNF plasma levels were observed in chronic phase (CP) and progressed CML and were significantly elevated in bone marrow and spleen in CML mouse models. Targeting the TNF-induced NFᴋB-signaling by overexpressing the inhibitory IᴋBα protein, impaired leukemogenesis in a retroviral model of Bcr-Abl1 driven disease.
Aims
Here, we aimed to complement TKI therapy by TNF pathway-directed treatment, to specifically target persisting CML cells in CP- and blast crisis (BC) CML.
Methods
Publicly available microarray data were analyzed for induction of TNF regulated pathways. 32D Bcr-Abl1, SCLtTA/Bcr-Abl1-BM, human K562 & KCL22 (imatinib-sensitive (S) and -resistant (R)) cells were evaluated using qRT-PCR, Western blot, annexin V & CFSE staining. CP-CML-CD34+-cells and BC mononuclear cells (MNCs) were subjected to colony-forming unit (CFU) assays or annexin V + CFSE measurements upon treatment with the IKK2 small molecule inhibitor LY2409881 (LY) +/- nilotinib or DMSO as control, in the presence of TNF, IL1α or IL1β.
Results
Analysis of microarray data unveiled enrichment of TNF signaling molecules in the CD34+CD38- compartment of CP-CML patient cells. 32D Bcr-Abl1 and primary murine SCLtTA Bcr-Abl1-BM cells exhibited enhanced TNF signaling compared to their Bcr-Abl1 negative counterparts. 32D Bcr-Abl1, KCL22 and K562 cells displayed no reduction in TNF signaling molecules upon TKI treatment, suggesting a Bcr-Abl1 kinase-independent persistence. The combination of nilotinib and LY reduced expression of IᴋBα and NFᴋB and abrogated their phosphorylation. This treatment reduced proliferation and induced apoptosis (fold change [fc]: 2.4; p<0.01) in KCL22 S cells. In primary CD34+ CP-CML cells, we discovered diminished clonogenic potential compared to TKI alone (fc: 0.05; p<0.01). Replating revealed nilotinib to preserve cells with colony-forming ability, as previously reported. These LSCs were in turn targeted by IKK2 inhibition (fc: 0.17; p<0.01). Reduction of the clonogenic potential was accompanied by apoptosis induction upon combinatorial treatment compared to TKI alone (fc: 2.5; p<0.01), also in the non-dividing cell fraction (fc: 5.2; p<0.001). Gene set enrichment of microarray data comparing BC to CP patient cells displayed further upregulation of TNF signaling in BC-LSCs. Interestingly, IKK2 inhibition could bypass TKI resistance in KCL22 R cells by apoptosis induction (fc: 2.4; p<0.01). Also, in BC-CML MNCs, combined nilotinib and LY treatment significantly decreased self-renewal (fc: 0.1; p<0.01) and induced apoptosis (fc: 1.8; p<0.01). Furthermore, combinatorial treatment targeted persistent CML-CP LSCs in the presence of IL1α and IL1β (fc: 0.6 & 0.5; p<0.05), underlining the relevance of IKK2 inhibition as global approach to target the pro-leukemic inflammatory environment.
Conclusion
In conclusion, we demonstrate that elevated TNF signaling in CML patients persists during TKI treatment. By combining IKK2 inhibition with a Bcr-Abl1-directed TKI, we were able to target non-dividing CP-CML LSCs but also TKI resistant BC-CML cells. We suggest targeting of TNF-induced signaling as a promising approach to address TKI persistence and resistance in CP- and BC-CML.
Keyword(s): Blast crisis, Chronic myeloid leukemia, Drug resistance, Tumor necrosis factor (TNF)