Immunomodulatory drugs (IMiDs) are a class of drugs, derived from the teratogenic compound thalidomide, that have made a major impact on treatment of many diseases, from multiple myeloma to assorted inflammatory diseases. From its dark beginnings as a teratogenic agent that caused phocomelia in newborn infants, thalidomide has resurged decades later as a potent immunomodulatory agent with widespread anti-inflammatory and anti-tumor effects. Research examining Thalidomide’s effects in vitro on malignant myeloma cells has led to the development of newer analogs, lenalidomide and pomalidomide, both of which are now available on the market. Clinically, these drugs have had a tremendous impact on patient progression-free survival, especially when administered in conjunction with other novel agents. Despite the numerous properties that have been reported for IMiDs, until recently, little was known about their mechanism of action. Knowledge of likely only one of IMiDs’ direct mechanism of action has not only opened up opportunities for additional discoveries, but also propelled research to better characterize genetic profiles of multiple myeloma patients and potential biomarkers of disease progression and response to treatment. This thesis will attempt to review the history and literature behind the biological mechanisms of IMiDs, the clinical risks and benefits of using such drugs as treatment for cancer, and future directions for areas of research.

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Class of 2012 Abstract / Specific Aims: Autologous stem cell transplant (ASCT) is the current gold standard following induction therapy to improve survival of multiple myeloma (MM). Lenalidomide (LEN) is used for treatment of MM before ASCT, but exposure may impair autologous peripheral blood stem cell (PBSC) mobilization. Chemomobilization with cyclophosphamide (CTX) has not been evaluated in this setting. CTX + filgrastim was investigated to determine if LEN-associated mobilization impairment can be abrogated. Methods: 36 pts (group A=12 pts who received ≥2 cycles of LEN and group B=24 pts without LEN) were analyzed retrospectively. Baseline characteristics were matched (p>0.05 for all variables). All pts received CTX (median group B, 1.5g/m2; median group A, 3gm/m2(p=0.18)) and filgrastim 10μg/kg/day. Primary outcomes include number of CD34+ cells collected and number of leukapheresis sessions. Secondary outcomes include failure to collect CD34+ cells and total CD34+ cells collected after second leukapheresis. Main Results: Total median number of CD34+ cells collected in group B=9.15x106/kg CD34+ cells and group A=7.43x106/kg CD34+ cells (p=0.159). Median number of apheresis sessions in group B=2 and group A=3 (p=0.42). Two of 12 pts with antecedent LEN usage failed to collect while no patient without previous LEN exposure failed to collect (p=0.105). Total number of CD34+ cells collected after 2 apheresis sessions for group B=8.13x106/kg CD34+ cells and group A=3.34x106/kg CD34+ cells (p=0.06). Conclusions: Chemomobilization with CTX + filgrastim yields robust PBSC collections irrespective of antecedent lenalidomide. There was a trend towards lesser PBSC collection in LEN-treated pts.

Mantle cell lymphoma (MCL) is a rare B cell neoplasm that accounts for approximately 4-8% of non-Hodgkin’s lymphomas (NHLs). The median age at diagnosis is 65 years with a male to female predominance of 3:1. It has also been demonstrated that female MCL patients have a greater response to therapy, especially immunomodulatory therapy compared to male MCL patients. The concept of cancer immunosurveillance is well described and it is perceived that females mount a greater immune response compared to males. In addition, although lymphomas are generally not perceived to be hormone controlled, epidemiological studies have demonstrated lower prevalence of lymphoma in females taking exogenous oestrogen. This aim of this thesis was to explore the gender difference observed in MCL. The study investigated the difference in the quantity of immune cells in the peripheral blood and lymph node biopsies of untreated male and female MCL patients. There was a significantly greater number of T cells in the peripheral blood of male MCL patients compared to the female MCL patients. Conversely, greater numbers of immune cells were observed in the lymph node biopsies of female MCL patients compared to male MCL patients. In addition, four NK cell activating receptors; NKp46, NKp44, NKp30 and NKG2D were examined to determine if their expression was different between the genders. The cell mediated cytotoxic function of the immune cells (PBMCs) from male and female MCL patients and healthy controls was also examined. Interestingly the healthy controls exhibited greater cytotoxicity compared to the MCL patients. PBMCs were incubated with oestrone (female hormone in postmenopausal women), lenalidomide and IL-2 to further investigate the effects of these on the immune cells from male and female MCL patients. Incubation with IL-2 resulted in a significant increase in the cytotoxicity activity of male MCL patients but not female MCL patients in this cohort. The lymph node biopsies from MCL patients were examined for the presence of oestrogen receptors. Oestrogen receptor β was predominantly expressed on MCL cells in all the biopsies examined. This is an area that warrants further studies. This thesis provides some insight into the mechanisms that may influence the gender difference observed in MCL, however further studies are needed.

Multiple Myeloma is cancer of plasma cells and is known to be highly invasive. Multiple Myeloma makes up 1% of cancer diagnosis in western countries and affects men more predominantly than women. The American Cancer Association estimates that 32,110 new cases will be diagnosed in the United States in 2019. Lenalidomide is one of the main therapies used for multiple myeloma patients, but it has toxic side effects such as thrombocytopenia, neutropenia, and anemia. The purpose of the study is to investigate new cytotoxic agents for the treatment of multiple myeloma. In addition to lenalidomide alone, this study examined the effects of doxycycline alone and in combination with lenalidomide. Lenalidomide cell cultures were treated at concentrations from 0.5μM to 10μM on untreated 24 well plates and doxycycline concentration ranging from 10μM-80μM. Following incubation, cell viability was tested using MTT assay and the samples were analyzed using spectrophotometry. When compared to lenalidomide, doxycycline monotherapy showed a greater decrease in overall cell viability in preliminary results. Our results show that there is benefit of using 10μM of Doxycycline at higher concentration of 5μM and 10μM of lenalidomide. The potential decrease in the concentration of lenalidomide used by adding doxycycline, may reduce the toxic side effects of lenalidomide. Further studies are necessary to confirm these preliminary results and investigate the mechanism of action in order to determine optimal combinations of these drugs.

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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.

Class of 2012 Abstract / Specific Aims: Autologous stem cell transplant (ASCT) is the current gold standard following induction therapy to improve survival of multiple myeloma (MM). Lenalidomide (LEN) is used for treatment of MM before ASCT, but exposure may impair autologous peripheral blood stem cell (PBSC) mobilization. Chemomobilization with cyclophosphamide (CTX) has not been evaluated in this setting. CTX + filgrastim was investigated to determine if LEN-associated mobilization impairment can be abrogated. Methods: 36 pts (group A=12 pts who received ≥2 cycles of LEN and group B=24 pts without LEN) were analyzed retrospectively. Baseline characteristics were matched (p>0.05 for all variables). All pts received CTX (median group B, 1.5g/m2; median group A, 3gm/m2(p=0.18)) and filgrastim 10µg/kg/day. Primary outcomes include number of CD34+ cells collected and number of leukapheresis sessions. Secondary outcomes include failure to collect CD34+ cells and total CD34+ cells collected after second leukapheresis. Main Results: Total median number of CD34+ cells collected in group B=9.15x106/kg CD34+ cells and group A=7.43x106/kg CD34+ cells (p=0.159). Median number of apheresis sessions in group B=2 and group A=3 (p=0.42). Two of 12 pts with antecedent LEN usage failed to collect while no patient without previous LEN exposure failed to collect (p=0.105). Total number of CD34+ cells collected after 2 apheresis sessions for group B=8.13x106/kg CD34+ cells and group A=3.34x106/kg CD34+ cells (p=0.06). Conclusions: Chemomobilization with CTX + filgrastim yields robust PBSC collections irrespective of antecedent lenalidomide. There was a trend towards lesser PBSC collection in LEN-treated pts.