Modulating Mitofusins to Enhance Therapeutic T Cell Expansion and Persistence Open Access
Chen, Kevin (Summer 2022)
Abstract
Chronic lymphocytic leukemia (CLL), a cancer of B-lymphocytes, is the most common leukemia in adults. While current frontline therapies for CLL, such as ibrutinib or a combination of venetoclax and obinutuzumab, have significantly improved clinical outcomes for patients with treatment-naïve CLL and relapsed and refractory CLL (RR-CLL), complete response (CR) rates for RR-CLL patients on ibrutinib remain between 5-14%. However, with the advent of chimeric antigen receptor T cells (CART), CR rates for RR-CLL are increased to around 26-29%, which is in sharp contrast to the 70-93% CR rates achieved using CART to treat patients with B-cell acute lymphoblastic leukemia (B-ALL). This discrepancy in response rates is due, in part, to the inherently immunosuppressive nature of CLL. In addition, T cells from CLL patients are significantly deficient in CD8 co-receptor expressing (CD8+) T cells, including naïve T (Tn), stem cell-like memory T (Tscm), and central memory T (Tcm) cells. Thus, elucidating translatable mechanisms for selective expansion of Tn, Tscm and Tcm from CLL patients is needed to improve the efficacy of CART cell therapy for CLL patients.
To that end, members of our lab have demonstrated that dual inhibition of Phosphoinositide 3-Kinase (PI3K) d/g isoforms with IPI-145 (duvelisib) preferentially expands CD8+ T cells, including Tn, Tscm and Tcm, as well as improves the in vivo persistence and cytotoxicity of CD19-targeted CART (CD19-CART). Furthermore, my immunoblot analysis of T cells cultured with duvelisib for 15 days reveals increases in the expression of epigenetic and transcriptional regulators of memory T cell programs, including sirtuins 1/3/5, FOXO1/3, TCF1/7, and ID3. In addition, ex vivo duvelisib treatment of CLL patient T cells increased expression of essential mitochondrial fusion proteins, mitofusins 1 and 2 (MFN1/2), and decreased serine 637 phosphorylation, and thereby inactivation, of mitochondrial fission protein, DRP1, both consistent with an increase in mitochondrial fusion. Interestingly, duvelisib increased the spare respiratory capacity of CD8+ T cells but did not alter the average mitochondrial cross-sectional area of bulk T cells, assessed by extracellular flux analysis and transmission electron microscopy (TEM), respectively. These data, taken together, demonstrate intersections between PI3K d/g inhibition, mitochondrial fusion, and T cell memory. However, as modulating PI3K signaling affects many downstream processes in T cell biology, duvelisib may not increase T cell expansion uniformly across all CLL patient samples.
Given the potential role of mitochondrial fusion in enhancing Tn/Tscm and Tcm persistence and function, I devised an alternative approach to PI3K d/g dual inhibition to induce mitochondrial fusion pharmacologically. Using the first-in-class mitofusin activating small molecule, MASM7, I sought to enhance MFN1/2 activity in CLL patient T cells. My data demonstrate that T cell cultures treated with MASM7 doses between 100nM - 250nM daily for nine days have >50% more CD8+ and CD8+CD27+CD45RO- T cells, compared to duvelisib and vehicle control groups. In addition, MASM7 induces significant increases in mitochondrial volume per mitochondrion and mitochondrial membrane potential to mass ratios, indicative of enhanced mitochondrial fusion. Interestingly, T cells treated with MASM7 do not exhibit increased MFN1/2 expression. However, further studies are required to confirm suggested changes in expression of biomarkers for T cell activation (TIM-3, PD-1, and LAG-3), proliferation (Ki-67), and self-renewal (TCF1).
In conclusion, I have shown that either dual inhibition of Phosphoinositide 3-Kinase (PI3K)d/g with IPI-145 (duvelisib) or MFN1/2 agonism with MASM7 improved the ex vivo expansion of CLL patient T cells. Further characterization of both strategies is required to confirm the cytotoxicity of MASM7-expanded CART, more definitively establish the mechanisms of action for duvelisib and MASM7, and determine potential synergy between duvelisib and MASM7 during ex vivo T cell expansion and CART generation, with predicted improved outcomes among CLL patients.
Table of Contents
Introduction
The therapeutic value of cart cell therapy in hematological malignancies
1
Discrepancies in response to cart therapy
1-2
Inhibition of PI3K d/g isoforms in the in vivo persistence and cytotoxicity of CD19-CART
2
Mitochondrial fusion and OXPHOS are required for the maintenance of T cell memory
3
Molecular structure of Mitofusin 1 and 2
3-4
Mitochondrial dynamics in cellular homeostasis and disease
4-5
MASM7 is a first-in-class direct activator of MFN1/2
5-9
Research Summary
10-12
Results
Inhibition of PI3K d/g favors mitofusin expression and activity
13-15
Electron imaging suggests inhibition of PI3K d/g favors fusion
16-17
Differences in mitochondrial quality between patients may predict response to duvelisib
17-25
Duvelisib induced pathways converge on MFN1/2
25-30
MASM7 promotes CD8+ T cell expansion and mitochondrial fusion
30-34
Discussion 35-36
Closing Remarks 37
Materials and Methods
Materials
38-41
Sample Collection, Leukapheresis, and PBMC isolation
42
T-cell activation and expansion
42-43
CART experiments
43
Flow Cytometry
43-44
Seahorse Assay
44
Immunoblot/Western Blot
44-45
Immunofluorescent Staining for Widefield Imaging
45-46
Statistical analysis
46
Protocol 1: Mitochondrial Mass and Membrane Potential Staining for Human Lymphocytes
47
Protocol 2: Immunoblot/Western Blot Protocol
48-49
Works Cited 50-54
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