Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Pearlie K. Epling-Burnette

Keywords

anti-tumor immunity, CD28, IMiDs, lenalidomide, Myelodysplastic Syndrome, PP2A

Abstract

T-cells are lymphocytes that make up part of the adaptive arm of the immune system, and are essential for efficient protection from and eradication of viruses and pathogens. T-cells not only play an important role in protection from external agents, but also regulate and prevent activation towards self-peptides and detect and remove erratically growing cells. Alterations in T-cell activation and suppression contribute to auto-immunity, immunocompromised disorders, and cancer progression.

The immune system, and T-cells in particular, provides daily surveillance, recognition and destruction of aberrant cells. Although the immune system is proficient at suppressing malignant progression, tumor cells acquire various methods of immune evasion. Myelodysplastic Syndrome (MDS) is a pre-malignant dysplastic disorder of the bone marrow characterized by ineffective hematopoiesis and clonality in the myeloid lineage, where lack of immune response has been implicated in the propensity for progression to acute myeloid leukemia (AML). Leukemia progression is associated with the acquisition of complex genetic abnormalities. Alterations in immune system regulation have been implicated in various stages of the disease process, although the role of the immune system in response to several therapies in MDS has not been fully discovered.

Lenalidomide is a small molecule therapeutic preferentially effective in MDS patients with an interstitial chromosome 5q deletion (del(5q)). Improved erythropoiesis has also been reported to occur in 20-30% of low-risk, non-del(5q) patients. Although lenalidomide is a potent immunomodulatory drug that potentiates T-cell and NK-cell responses, the T-cell compartment in MDS is highly deregulated by aberrant repertoire skewing, decreased function and abnormal naïve and memory cell homeostasis. The presence of lymphoid infiltrates in the bone marrow of lenalidomide-responsive patients suggests that T-cells may participate in the hematopoietic response, but it is unclear if lenalidomide is capable of reversing these functional T-cell defects. We therefore assessed immunological changes in low-risk MDS patients before and after 16-weeks of lenalidomide therapy, and assessed the relationship to erythroid response. Although MDS T-cells were anergic prior to treatment, we have shown that T-cells in responders have a significant increase in antigen-induced proliferative response and T helper type-1 (Th1) cytokine production (IL-2, IFN-γ, TNF-α) compared to non-responders. The change in function positively correlated with an increase in naïve T-cells and a decrease in memory cells, indicating that lenalidomide has immunomodulatory activity to reverse anergy in MDS.

Although it is known that lenalidomide may increase T-cell activation and proliferation in the absence of co-stimulatory signals, a direct mechanism of action has yet to be elucidated. Since CD28 is one of the most important co-stimulatory molecules deregulated in cancer, we therefore set out to determine if the expression of CD28 was essential for lenalidomide's mechanism in T-cells. We knocked out CD28 expression in healthy donor T-cells, and sorted on inherently deficient, CD28null, T-cells that accumulate in older healthy donors and found that lenalidomide-induced proliferation and function were completely ablated within the CD28null subset. These data indicate the immunotyrosine-based activation motifs (ITAMs) on the intracellular domain of the CD28 receptor are necessary for lenalidomide action.

Interestingly, during the natural aging process, repeated exposure to antigens results in the accumulation of CD28null T-cells that are phenotypically distinct and functionally deficient due to excessive proliferative history in vivo. We therefore examined whether CD28 expression on MDS patient T-cells affected responses to lenalidomide, and if this could be used as a predictive biomarker of responsiveness. We found that patients who fail lenalidomide therapy had higher CD8+ Terminal Effector Memory (TEM), which are inherently CD28null, and that non-responders had an overall increase in CD4+ and CD8+CD28null T-cells, as well as an increase in CD28null cells within the TEM compartment compared to hematologic responders.

We then sought to determine the particular protein target of lenalidomide responsible for increased CD28 receptor signaling in T-cells. Several targets in a variety of cell types have been postulated, although the direct mechanism in T-cells is unclear. Our group has previously shown that lenalidomide inhibits the activity of two haplodeficient phosphatases located within the commonly deleted region (CDR) on chromosome 5q in the MDS myeloid clone, Protein Phosphatase 2A (PP2A) and Cdc25c. PP2Ac is known to bind CD28 and is hypothesized to inhibit T-cell co-stimulation. Therefore, it is plausible that lenalidomide and other IMiDs inhibit the phosphatase activity of PP2A which leads to increased activation of T-cell proximal signals dependent on CD28 expression. We examined this hypothesis using molecular modeling and virtual screening and found that all of the IMiDs (lenalidomide, pomalidomide, and thalidomide) can theoretically interact with the catalytic pocket of the PP2A heterotrimer, potentially inhibiting PP2Ac activity. In vitro phosphatase activity assays supported these findings as lenalidomide-inhibition of PP2Ac activity was seen in both ad293 and Jurkat cell lines, and in primary T-cells. Mutations of theorized lenalidomide hydrogen-bond sites within the catalytic pocket of PP2A rendered the enzyme catalytically dead, indicating that these are important residues for enzymatic activity, but unfortunately could not be used to determine if lenalidomide activity was disrupted by mutation of those sites.

These data together suggest that the ability of lenalidomide to augment immune activation in vivo in MDS patients, and potentially other diseases, is extremely important to patient response. Also, that CD28 expression on T-cells is essential for lenalidomide immune-mediated tumor eradication through CD28 downstream signaling, and potentially through inhibition of PP2A. These results are useful in designing future lenalidomide-combination therapy trials in other hematologic and solid malignancies, and could be used to help stratify patients for future therapeutic decisions in MDS and other malignancies.

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