Assessment of The Epichaperome In Acute Myeloid Leukemia Lisa - - PowerPoint PPT Presentation

Assessment of The Epichaperome In Acute Myeloid Leukemia

Lisa Toudic

Acute Myeloid Leukemia

• 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

AML can develop from either of these cells by acquisition of mutation(s)

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 epichaperome

HSP90

• 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

folding chaperones called the epichaperome

HSP90 Chaperone

• All cells have

chaperones

• Fold proteins in cells

to organize them

• Necessary for cell

survival

Guzman and Chiosis, 2016

• Discovered a protein-complex
• f 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

possible flow cytometry assay to determine if cells had the epichaperome, using PU-FITC probe. Positive Epichaperome Cell PU-FITC Compound

• 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

possible flow cytometry assay to determine if cells had the epichaperome, using PU-FITC probe. Negative Epichaperome Cell PU-FITC Compound

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 Binding Assay

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 PUH71

• 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

Guzman and Chiosis (2016) - Novel PUH71 Epichaperome Inhibitor

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

2 1

Novel Binding Assay Novel Viability 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

• Unstained
• Stained with

PU-FITC (epichaperome probe)

• Stained with FITC9

(background control)

UT PU-FITC FITC9

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 wells

• Untreated
• Treated with

0.5µM of PUH71 (epichaperom e inhibitor)

US 0.5µM PUH71

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 debris Live vs. Dead cells

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

2 1

Novel Binding Assay Novel Viability Assay

Binding Assay Results

Cell Line Controls

Viability Assay Results

Samples Used From Binding Assay

Cell Line Controls

Control

Conclusion Layout

2 1

Novel Binding Assay Novel Viability 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

This figure can only be utilized for the purposes of this paper, photography or publishing on any media is strongly prohibited.

Future Research

In the future...

• Test for long-term effects of the treatment
• Analyze for any potential recurrent resistant cells after the

PUH71 treatment

• Try to find if other cancers where these assays can be

utilized

• Reduce toxicity levels in the PUH71 treatment
• Test results of samples who aren’t highly positive

epichaperome or highly negative epichaperome

• Analyze the “Middle Ground”

References

Bartkowiak, K., & Pantel, K. (2016, October 05). Cancer: A shocking protein complex. Retrieved September 30, 2018, from https://www.nature.com/articles/nature19476 Dahlmann, H. A. (2016, November 18). Chaperome Complexes Influence Tumor Survival. Retrieved September 30, 2018, from https://pubs.acs.org/doi/abs/10.1021/acschembio.6b00969?journalCode=acbcct Loh, M. L., Vattikuti, S., Schubbert, S., Reynolds, M. G., Carlson, E., Lieuw, K. H., . . . Shannon, K. M. (2004, March 15). Mutations in PTPN11 implicate the SHP-2 phosphatase in

• leukemogenesis. Retrieved September 30, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/14644997

Rodina, A., Wang, T., Yan, P., Gomes, E. D., Dunphy, M. P., Pillarsetty, N., . . . Chiosis, G. (2016, October 05). The epichaperome is an integrated chaperome network that facilitates tumour survival. Retrieved September 30, 2018, from https://www.nature.com/articles/nature19807 Tyner, J. W., Tognon, C. E., Bottomly, D., Wilmot, B., Kurtz, S. E., Savage, S. L., . . . Druker, B. J. (2018, October 17). Functional genomic landscape of acute myeloid leukaemia. Retrieved November 13, 2018, from https://www.nature.com/articles/s41586-018-0623-z?_ga=2.125937806.2841101.1540771200-2083207147.1540771200 Zong, H., Gozman, A., Caldas-Lopes, E., Taldone, T., Sturgill, E., Brennan, S., . . . Guzman, M. L. (2015, December 15). A Hyperactive Signalosome in Acute Myeloid Leukemia Drives Addiction to a Tumor-Specific Hsp90 Species. Retrieved September 30, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/26628369 Borman, S. (n.d.). Heat-shock protein complexes serve as cancer drug targets. Retrieved November 25, 2018, from https://cen.acs.org/articles/94/i40/Heat-shock-protein-complexes-serve.html Acute myeloid leukemia. (2018, November 07). Retrieved November 25, 2018, from https://en.wikipedia.org/wiki/Acute_myeloid_leukemia

• G. (n.d.). MYC Gene(Protein Coding) MYC Proto-Oncogene, BHLH Transcription Factor. Retrieved

December 9, 2018, from https://www.genecards.org/cgi-bin/carddisp.pl?gene=MYC Stein, Eytan M., et al. “Molecular Remission and Response Patterns in Patients with Mutant-IDH2 Acute Myeloid Leukemia Treated with Enasidenib.” Blood Journal, American Society of Hematology, 14 Feb. 2019, www.bloodjournal.org/content/133/7/676. Speranza, G., Anderson, L., Chen, A. P., Do, K., Eugeni, M., Weil, M., . . . Kummar, S. (2018, April). First-in-human study of the epichaperome inhibitor PU-H71: Clinical results and metabolic profile. Retrieved March 12, 2019, from https://www.ncbi.nlm.nih.gov/pubmed/28808818

Assessment of The Epichaperome In Acute Myeloid Leukemia

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