A SECOND GENERATION LYSOSOMOTROPIC AGENT DRIVES LEUKAEMIC STEM CELL DIFFERENTIATION AND SENSITIZES THEM TO TYROSINE KINASE INHIBITOR TREATMENT IN VITRO AND IN VIVO
(Abstract release date: 05/18/17)
EHA Library. Baquero P. 06/24/17; 181768; S481
Pablo Baquero
Contributions
Contributions
Abstract
Abstract: S481
Type: Oral Presentation
Presentation during EHA22: On Saturday, June 24, 2017 from 16:00 - 16:15
Location: Room N101
Background
Autophagy is a conserved catabolic process that delivers cytoplasmic constituents to the lysosomes. We have previously shown that the lysosomotropic agent hydroxychloroquine (HCQ) inhibits autophagy and sensitizes Chronic Myeloid Leukaemia (CML) stem cells (LSCs) to tyrosine kinase inhibitors (TKIs) treatment. However, the biological effects of autophagy inhibition in LSCs in vivo are currently unknown and remain to be investigated. Furthermore, recent clinical studies showed that maximum tolerated dose of HCQ does not achieve consistent autophagy inhibition in cancer patients. Therefore further pre-clinical studies using more potent 2nd generation lysosomotropic agents, alone and in combination with TKIs, are vital.
Aims
Here we aim to investigate the functional effects of autophagy inhibition in LSCs both in vitro and in vivo using the highly potent lysosomotropic agent Lys05. Additionally, we aim to address whether Lys05 achieves autophagy inhibition in the most primitive LSC populations in vivo and whether it targets LSCs more effectively than HCQ when combined with TKIs.
Methods
In this study, we used primary stem-cell enriched samples (CD34+ cells) derived from CML patients at diagnosis. For in vivo studies, we used a human patient-derived xenograft (PDX) model and an inducible transgenic CML model in which the expression of BCR-ABL is induced at a stem/progenitor level (Scl-tTa-BCR-ABL). To accurately measure autophagy flow in long term LSCs in vivo, we generated the transgenic mouse Scl-tTa-BCR-ABL/GFP-LC3 by crossing the Scl-tTa-BCR-ABL model with a mouse bearing the autophagy marker LC3 fused to GFP.
Results
Firstly, we show that Lys05 targets LSCs more potently than HCQ in vitro by achieving a 60% and a 35% reduction in number of CD34+CD38- and CFSEmax/CD34+CD133+ cells respectively. Interestingly, Lys05 promoted a 40% loss of quiescent cells and induced myeloid differentiation of CD34+ cells. Functional long-term culture initiating cell (LTC-IC) assay demonstrated that, while HCQ had moderate effects, Lys05 decreased the number of LSC-derived colonies by 80%.
Additionally, we show that Lys05 inhibits autophagy flow more efficiently than HCQ both in the Scl-tTa-BCR-ABL/GFP-LC3 model and in patient-derived progenitor cells. Analysis of bone marrow (BM) cells from Lys05-treated leukaemic mice (but not from HCQ-treated mice), showed a statistically significant 35% decrease (p=0.0469) in the most primitive population Lin-Sca+c-kit+CD48-CD150+ followed by a 50% increase (p=0.0231) of progenitors Lin-Sca+c-kit+. This result indicates differentiation of LSCs towards a more progenitor phenotype following potent autophagy inhibition.
Finally, to test the in vivo effect of Lys05 on the most primitive human LSCs we transplanted CD34+ CML cells into irradiated NSG mice. Remarkably, using this PDX model we show that while 3 weeks in vivo treatment with HCQ had no effects when combined with TKIs, Lys05 and TKI treatment nearly eliminated engrafted primitive Philadelphia positive CD34+CD38- and CD34+CD133+ cells.
Conclusion
Overall, we demonstrate that lysosomal inhibition induces loss of quiescence and drives differentiation of LSCs in vitro and in vivo. Furthermore, our results show that Lys05 achieves autophagy inhibition in LSCs and effectively sensitizes LSCs to TKIs in vitro and in vivo. Therefore, 2nd generation lysosomotropic agents should be considered as a potential alternative to HCQ in order to eliminate LSCs and achieve cure for CML patients.
Session topic: 7. Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Murine models, Leukemic Stem Cell, Differentiation
Abstract: S481
Type: Oral Presentation
Presentation during EHA22: On Saturday, June 24, 2017 from 16:00 - 16:15
Location: Room N101
Background
Autophagy is a conserved catabolic process that delivers cytoplasmic constituents to the lysosomes. We have previously shown that the lysosomotropic agent hydroxychloroquine (HCQ) inhibits autophagy and sensitizes Chronic Myeloid Leukaemia (CML) stem cells (LSCs) to tyrosine kinase inhibitors (TKIs) treatment. However, the biological effects of autophagy inhibition in LSCs in vivo are currently unknown and remain to be investigated. Furthermore, recent clinical studies showed that maximum tolerated dose of HCQ does not achieve consistent autophagy inhibition in cancer patients. Therefore further pre-clinical studies using more potent 2nd generation lysosomotropic agents, alone and in combination with TKIs, are vital.
Aims
Here we aim to investigate the functional effects of autophagy inhibition in LSCs both in vitro and in vivo using the highly potent lysosomotropic agent Lys05. Additionally, we aim to address whether Lys05 achieves autophagy inhibition in the most primitive LSC populations in vivo and whether it targets LSCs more effectively than HCQ when combined with TKIs.
Methods
In this study, we used primary stem-cell enriched samples (CD34+ cells) derived from CML patients at diagnosis. For in vivo studies, we used a human patient-derived xenograft (PDX) model and an inducible transgenic CML model in which the expression of BCR-ABL is induced at a stem/progenitor level (Scl-tTa-BCR-ABL). To accurately measure autophagy flow in long term LSCs in vivo, we generated the transgenic mouse Scl-tTa-BCR-ABL/GFP-LC3 by crossing the Scl-tTa-BCR-ABL model with a mouse bearing the autophagy marker LC3 fused to GFP.
Results
Firstly, we show that Lys05 targets LSCs more potently than HCQ in vitro by achieving a 60% and a 35% reduction in number of CD34+CD38- and CFSEmax/CD34+CD133+ cells respectively. Interestingly, Lys05 promoted a 40% loss of quiescent cells and induced myeloid differentiation of CD34+ cells. Functional long-term culture initiating cell (LTC-IC) assay demonstrated that, while HCQ had moderate effects, Lys05 decreased the number of LSC-derived colonies by 80%.
Additionally, we show that Lys05 inhibits autophagy flow more efficiently than HCQ both in the Scl-tTa-BCR-ABL/GFP-LC3 model and in patient-derived progenitor cells. Analysis of bone marrow (BM) cells from Lys05-treated leukaemic mice (but not from HCQ-treated mice), showed a statistically significant 35% decrease (p=0.0469) in the most primitive population Lin-Sca+c-kit+CD48-CD150+ followed by a 50% increase (p=0.0231) of progenitors Lin-Sca+c-kit+. This result indicates differentiation of LSCs towards a more progenitor phenotype following potent autophagy inhibition.
Finally, to test the in vivo effect of Lys05 on the most primitive human LSCs we transplanted CD34+ CML cells into irradiated NSG mice. Remarkably, using this PDX model we show that while 3 weeks in vivo treatment with HCQ had no effects when combined with TKIs, Lys05 and TKI treatment nearly eliminated engrafted primitive Philadelphia positive CD34+CD38- and CD34+CD133+ cells.
Conclusion
Overall, we demonstrate that lysosomal inhibition induces loss of quiescence and drives differentiation of LSCs in vitro and in vivo. Furthermore, our results show that Lys05 achieves autophagy inhibition in LSCs and effectively sensitizes LSCs to TKIs in vitro and in vivo. Therefore, 2nd generation lysosomotropic agents should be considered as a potential alternative to HCQ in order to eliminate LSCs and achieve cure for CML patients.
Session topic: 7. Chronic myeloid leukemia - Biology
Keyword(s): Chronic myeloid leukemia, Murine models, Leukemic Stem Cell, Differentiation
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