Multiple myeloma (MM) is an incurable B cell malignancy that is characterized by growth and survival of neoplastic plasma cells within the bone marrow microenvironment. Despite progress achieved in the treatment of MM, including the use of high-dose chemotherapy and autologous stem cell transplantation, a considerable proportion of patients are refractory to the therapies. This variability in response is likely related cellular morphology, molecular genetics and functional heterogeneity within the MM microenvironment. Our previous studies using PKH67 fluorescent tracers showed that MM heterogeneity is correlated with the presence of quiescent MM cells. We isolated quiescent MM cells near purity on the basis of their ability to retain the lipophilic dye, PKH67, as a consequence of their quiescent nature. We also allowed MM cells to cycle in mice instead of in vitro, which reflects their nature within the microenvironment. Our study is the first to reveal a quiescent MM cell niche and the effects of functional interactions between MM stem-like cells and microenvironment. After cycling in vivo, rare quiescent PKH+ cells preferentially reside within osteoblastic niches, rather than in vascular niches of the bone marrow or spleen. Functional analyses of PKH+ cells from osteoblastic niches revealed enhanced stem-like properties in vitro by increasing colony forming units. In addition, these PKH+ cells were highly tumorigenic, as compared to PKH- cells, and were resistant to a variety of clinically relevant chemotherapeutic drugs. cDNA microarray analyses revealed that a tripartite motif protein, TRIM44, is highly upregulated in PKH+ cells from the osteoblastic niche. TRIM family proteins are involved in a broad range of biological processes and alterations in expression and function are associated with pathologies such as birth defects, neurodegenerative diseases, viral infections and cancer. Unlike other TRIM family member proteins, TRIM44 contains a zinc finger ubiquitin binding domain (ZF-UBP) in the N-terminal region instead of a RING domain. UBP domains are often found in the deubiquitinating enzymes containing ubiquitin specific peptidase. Collectively, these data suggest that TRIM44 may function as an ubiquitin specific peptidase-like TRIM and act as a cancer promoting gene regulating deubiquitination and/or stabilization of oncogenes. TRIM44 was first cloned in 2001 yet very little is known about its function. Interestingly, several reports have shown overexpression of TRIM44 in cancer, including 16% of epithelial cancers with amplified TRIM44 expression. Therefore, TRIM44 may serve as a potential cancer biomarker for MM. In this project, we will use biochemical and molecular approaches to delineate TRIM44 functions in quiescent MM cells. We hypothesize that TRIM44 is essential for maintenance of PKH+ MM cell quiescence within osteoblastic niches. Furthermore, we propose that TRIM44 confers stem-like functions to MM cells.
In Specific Aim 1, we will define TRIM44 functions in quiescent MM niches in vivo.
In Specific Aim 2, we will investigate roles of TRIM44 in autophagy formation and MM survival. In summary, completion of this project will deepen our knowledge of MM stem-like cells and their microenvironment and establish TRIM44 as a potential therapeutic target in MM.
Proposal Description: Multiple myeloma (MM) is the clonal expansion of neoplastic plasma B cells within the bone marrow and is the second most common hematologic malignancy in the United States. Functional and phenotypic heterogeneity within MM cells contribute drug resistance and therapy relapse. Our previous study showed that heterogeneity of MM cells is correlated with quiescent stem cell content. These quiescent MM stem-like cells preferred to reside osteoblastic niche of the bone rather than vascular or spleen niche. Moreover, these MM stem-like cells were highly tumorigenic upon transplantation in mice and drug resistant to clinically relevant drugs. In order to understand molecular pathways regulating therapy resistant MM stem cell characteristics, we performed gene profiling analyses. In this grant we will delineating functions of candidate genes using unique biochemical and molecular analyses. A better understanding of quiescent MM cells will help to understand drug resistant clones within MM.
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