Assessment of The Epichaperome In Acute Myeloid Leukemia Lisa Toudic
Acute Myeloid Leukemia AML can develop from either of these cells by acquisition of mutation(s) - Characterized by a rapid growth of these abnormal cells → build up in the bone marrow and blood - Interferes with normal blood cells - Mostly seen in older patients Acute myeloid leukemia. (2018, November 07). Retrieved November 25, 2018, from https://en.wikipedia.org/wiki/Acute_myeloid_leukemia
Treatment Options - Chemotherapy - Targeted drug therapy - Stem cell transplant - Not all methods are effective for patients with AML Why are these patients not responding to treatment? Levis, M. J., PhD. (n.d.). Rethinking the Treatment of Older Adults With Acute Myeloid Leukemia - ppt download. Retrieved April 20, 2019, from https://slideplayer.com/slide/5683214/
Guzman and Chiosis, 2016 - Discovered a protein-complex of folding chaperones called the HSP90 epichaperome - Heat Shock Proteins (HSPs) are seen in cells that are under a lot of stress (ie. treatment) - HSP90 is a HSP that was noticeably found in the epichaperome
Guzman and Chiosis, 2016 - Discovered a protein-complex of HSP90 folding chaperones called the epichaperome Chaperone - All cells have chaperones - Fold proteins in cells to organize them - Necessary for cell survival
Guzman and Chiosis, 2016 - Discovered a protein-complex of folding chaperones called the epichaperome - Epichaperome allows cancer inducing proteins to function by maintaining their proper shape - The epichaperome is not related to a specific gene, protein or mutation Borman, S. (2016, October 10). Heat-shock protein complexes serve as cancer drug targets. Retrieved April 21, 2019, from https://cen.acs.org/articles/94/i40/Heat-shock-protein-complexes-serve.html
Guzman and Chiosis (2016) - Novel Binding Assay - Concluded a PU-FITC possible flow Compound cytometry assay to determine if cells had the epichaperome, using PU-FITC probe. Positive Epichaperome Cell
Guzman and Chiosis (2016) - Novel Binding Assay - Concluded a possible flow cytometry assay to determine if cells had the epichaperome, using PU-FITC probe. Positive Epichaperome Cell after PU-FITC Binding
Guzman and Chiosis (2016) - Novel Binding Assay - Concluded a PU-FITC possible flow Compound cytometry assay to determine if cells had the epichaperome, using PU-FITC probe. Negative Epichaperome Cell
Guzman and Chiosis (2016) - Novel Binding Assay - Concluded a possible flow cytometry assay to determine if cells had the epichaperome, using PU-FITC probe. Negative Epichaperome Cell after Pu-FITC Binding
Guzman and Chiosis (2016) - Novel PUH71 Epichaperome Inhibitor - The epichaperome is reliant on PUH71 Heat Shock Proteins (HSPs) - It was inferred that if one HSP was eliminated the epichaperome would dissolve - Rodina et al., 2016 discovered a way to inhibit HSP90 through its ATP binding pocket with PUH71
Guzman and Chiosis (2016) - Novel PUH71 Epichaperome Inhibitor - The epichaperome is reliant on Heat Shock Proteins (HSPs) - It was inferred that if one HSP was eliminated the epichaperome would dissolve - Rodina et al., 2016 discovered a way to inhibit HSP90 through its ATP binding pocket with PUH71
Guzman and Chiosis (2016) - Novel PUH71 Epichaperome Inhibitor - The epichaperome is reliant on Heat Shock Proteins (HSPs) - It was inferred that if one HSP was eliminated the epichaperome would dissolve - Rodina et al., 2016 discovered a way to inhibit HSP90 through its ATP binding pocket with PUH71
Guzman and Chiosis (2016) - Novel PUH71 Epichaperome Inhibitor - The epichaperome is reliant on Heat Shock Proteins (HSPs) - It was inferred that if one HSP was eliminated the epichaperome would dissolve - Rodina et al., 2016 discovered a way to inhibit HSP90 through its ATP binding pocket with PUH71
Guzman and Chiosis (2016) - Novel PUH71 Epichaperome Inhibitor - The epichaperome is reliant on Heat Shock Proteins (HSPs) - It was inferred that if one HSP was eliminated the epichaperome would dissolve - Rodina et al., 2016 discovered a way to inhibit HSP90 through its ATP binding pocket with PUH71 Snayk. (2008, October 29). Dead cell. Retrieved April 21, 2019, from https://snayk.newgrounds.com/news/post/216741
Questions/Goals - To determine whether AML cells have the epichaperome by testing for its levels using a novel flow cytometry assay - Successfully eliminate cells that are reliant on the epichaperome using PUH71 inhibitor
Hypothesis - PU-FITC can be used to evaluate epichaperome levels in AML patient samples, and those cells will be most sensitive to epichaperome inhibitors - PUH71 targets HSP90, when it is part of the epichaperome, which eliminates it from the epichaperome and induces apoptosis in AML relapse refractory epichaperome positive samples
Method Layout 1 2 Novel Novel Binding Viability Assay Assay
Novel Binding Assay Methods
Primary Cells and Cell Line Controls - 11 primary (patient) samples were used - All had relapsed refractory AML - Unresponsive to prior treatment - 2 control cell lines were used - MV411 → positive (has) epichaperome - HL60 → negative (lacks) epichaperome
Staining of Samples - Every sample/cell line had triplicate wells UT - Unstained - Stained with PU-FITC PU-FITC (epichaperome probe) FITC9 - Stained with FITC9 (background control)
Flow Cytometry - Used to find epichaperome levels - Fluorescent compounds are visualized in the ( FITC channel ) in flow cytometry
Antibodies - Samples were also stained with antibodies that show different functions in flow cytometry
Gating of Cells - Used on the cells after flow cytometry - Gating → sectioning of the proper cells to analyze
FITC Channels
FITC Channels - The overlap between the two gives the Binding in that cell type - Binding → determines a unit which tells us if cells have the epichaperome Binding
FOLD Binding Calculations - Used counts from lymphocytes and blasts only - Blasts → AML cells - Lymphocytes → normal cells
FOLD Binding Calculations - Calculated the Mean Fluorescence Intensity (MFI) for the channels in each sample
FOLD Binding Calculations - Concluded by Guzman et al., 2016 that if FOLD binding had a value over 2 it had the epichaperome - If value was below 2, the epichaperome was absent
Novel Viability Assay Methods
Samples and Controls - Five of the samples tested in the Binding Assay were tested in the Viability Assay - Lymphocytes were used as a control
Plating Layout - Every sample/cell line had triplicate US wells - Untreated 0.5µM PUH71 - Treated with 0.5µM of PUH71 (epichaperom e inhibitor)
Flow Cytometry/Antibodies - Before running in Flow Cytometry they were stained with DAPI (Viability Dye) - Cells were stained with antibodies which show different functions on the machine
Gating of Cells - The same gating method was used as the Binding Assay - Separated from debris - Separated from live and dead cells Separated from Live vs. Dead cells debris
Viability Calculations - Analyzed percent decrease of Blasts from control (untreated samples) to the PUH71 treated samples - Calculated for a significant decrease from untreated cells and treated with 0.5 µM of PUH71
Results Layout 1 2 Novel Novel Binding Viability Assay Assay
Binding Assay Results
Cell Line Controls
Viability Assay Results
Samples Used From Binding Assay Cell Line Controls
Control
Conclusion Layout 1 2 Novel Novel Binding Viability Assay Assay
Binding Assay Conclusion
Key Findings Through PU-FITC Assay - The levels of PU-FITC fluorescence correlate to the presence of the epichaperome - Successful identification of the epichaperome with the PU-FITC assay opens new opportunities for ways to target the epichaperome and eliminate AML in patients - This assay is an example of the use of precision medicine in a successful manner - New approach from the tumor specific (mutations) methods of treatment of previous studies
Viability Assay Conclusion
Key Findings Through PUH71 Assay - The samples that were epichaperome positive responded to the PUH71 treatment effectively - This opens new possible ways of treating patients who have eipchaperome reliant cancer cells - If samples had a high abundance of PU-FITC they responded the best to treatment Results led to the use of PUH71 in a Compassionate Trial...
Compassionate Trial - The use of PUH71 was possible due to its approval in Speranza G, 2017 study when it was tested in tumor samples that were previously refractory to other treatments - Example of testing the PUH71 in vivo with the patient sample that was concluded to have high abundance of PU-FITC (positive epichaperome) through the Binding Assay
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Future Research
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