Glutamate-Cysteine Ligase Modifier Subunit Overexpression Confers Resistance Against Busulfan

Conference: ASGCT 23rd Annual Meeting

Project: Chemoprotection in Gene-Modified HSPC Transplants

Authors:
Serhat Gumrukcu
Phillip A Musikanth
Tung Nguyen

 

ABSTRACT

Increased expression GCL modifier subunit alone confers substantial chemoprotection against busulfan.

Seraph Research Institute · Translational Research

Glutamate-Cysteine Ligase Modifier Subunit Overexpression Confers Resistance Against Busulfan

Serhat Gümrükcü 1, Phillip A. Musikanth 1, Tung Nguyen 1,2

1 Seraph Research Institute, Los Angeles, CA; 2 Enochian Biosciences, Los Angeles, CA

Glutamate-Cysteine Ligase Modifier Subunit Overexpression Confers Resistance Against Busulfan

Serhat Gümrükcü 1, Phillip A. Musikanth 1, Tung Nguyen 1,2

1 Seraph Research Institute, Los Angeles, CA; 2 Enochian Biosciences, Los Angeles, CA

BACKGROUND

• Lack of sufficient engraftment in gene-modified hematopoietic

stem/progenitor cells (HSPCs) therapies has been the biggest

challenge.

•  Usage of chemo-resistance gene against cytotoxic agents to

select for these cells in vivo has been proposed.

• Clearance of busulfan -commonly used cytotoxic chemotherapy

agent for conditioning regimens in HSPC transplants- is

achieved by Glutathione (GSH) conjugation. Increased levels of

GSH in the cytoplasm mediates busulfan toxicity.

• The first step and rate-limiting enzyme of GSH synthesis

pathway involves glutamate-cysteine ligase (GCL)(Fig. 1).

• We evaluated whether increasing the GCL enzymatic activity

through overexpressing its modifier subunit GCLM would

confer protection against busulfan toxicity in vitro.

METHODS

• Jurkat, CEM, and TF-1a cell lines were transduced with a lentiviral vector

that expresses GCLM and green fluorescent protein (GFP) genes (Fig. 2A).

• Chemoprotection experiments were performed by incubating

untransduced or mixed populations of (20% transduced, 80% untransduced) cells with busulfan in concentrations ranging from low (2 to 10 ug/ml) to moderate and high (10 to 200 ug/ml) for 1 or 2 hours. Cells were then incubated and viable cell counts were documented in

busulfan-free culture for the 24, 48, 72 and 96-hr timepoints (Fig. 2B).

•  Pre- and post-transduction, and post-exposure GSH levels were measured with fluorometric assays by FACS analysis.

• Cell survival and proliferation were measured daily by Nucleocounter NC- 200 and flow cytometry using PI and 7-AAD staining protocols. The Chemoselection

RESULTS

• GSH level increased in transduced Jurkat, CEM and TF-1a cells (Fig. 3A). There was no effect on cell viability and proliferation due to the overexpression of GCLM (Fig. 3B)

• GSH levels in transduced cells remained stable throughout the experiment, whereas GSH in untransduced cells was depleted after busulfan exposure (Fig. 4)

• Increased GSH activity in GLCM-transduced-CEM cells rendered at least 3.5-fold protection against busulfan at lower doses (Fig. 5)

• At higher doses (10 or 20 ug/ml) of busulfan exposure, it also exhibited protection and proliferation of the transduced cell populations (Fig. 6)

• Moderate levels of busulfan exposure achieved selection of

chemo-protected cells and 100% transduced cell population in 72- 96h (Fig. 7)

DISCUSSION

Increased expression GCL modifier subunit alone confers substantial chemoprotection against

busulfan at multiple varying doses and times of exposure.

• This indicates that GCLM transgene expression could be used for busulfan protection, hence

selection of genetically modified cells.

• To investigate the potential usage of GCLM overexpression in clinical applications, in vitro

chemo-protection experiments are to be done in primary human CD34+ HSPCs.

• Following in vitro experiments on primary human CD34+ HSPCs, syngeneic or humanized mouse models will be used for in vivo Proof-of-Concept.

CONCLUSION

One of the biggest obstacles in gene-modified autologous HSPC transplants is lack of sufficient

engraftment.

•  Busulfan is an agent that has higher toxicity on bone marrow stem cells and limited toxicity to

other organs.

•  In gene-modified autologous non-myeloablative bone marrow transplants, GCLM gene could be utilized to increase the engraftment rate of gene-modified stem cells

Corresponding author Serhat Gumrukcu

Seraph Research Institute Los Angeles, CA

drs@seraphinstitute.org