Mantle cell lymphoma (MCL) is a rare but aggressive form of B cell non-Hodgkin lymphoma in which therapy resistance is common. New therapeutic options have extended survival in refractory MCL but have not provided durable remission. Tools are needed to assess the molecular and genetic changes associated with therapy resistance. Therefore, therapy-resistant MCL cell lines were established from the liver, kidney and lungs of human Granta 519-bearing NOD-SCID (non-obese diabetic-severe combined immunodeficiency) mice following treatment with CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy in combination with bortezomib. The cytomorphologies, immunophenotypes, growth patterns in semi-solid agar, cytogenetic profiles and gene expression differences between these cell lines were characterized to identify major changes associated with therapy resistance. Therapy-resistant cell lines exhibit more aggressive growth patterns and markedly different gene expression profiles compared to parental Granta 519 cells. Thus, these stable therapy-resistant cell lines are useful models to further study the molecular basis of drug resistance and to identify clinically relevant molecular targets in MCL.

Chronic Lymphocytic Leukemia (CLL), the most prevalent adult leukemia in western countries, which is highly heterogeneous with a very variable clinical outcome. Emerging evidence indicates that the stromal tumor microenvironment (STME) and stromal associated genes (SAG) play important roles in the pathogenesis and progression of CLL. However, the precise mechanisms by which STME and SAG are involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from lymph nodes (LN) (n=15), bone marrow (BM) (n=18), and peripheral blood (PB) (n=20). Interestingly, LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were found to be overexpressed while ITGB2, DLC1 and ITGA6 were under expressed in LN-CLL compared to BM-CLL and PB-CLL. This is suggestive of a role for LN-mediated TME in CLL cell survival/progression. Among these genes, expression of MYLK, CAV1 and CAV2 correlated with clinical outcome as determined by time to first treatment. Together, our studies show that members of the stromal signature, particularly in the CLL cells from lymph nodes, regulate CLL cell survival and proliferation and thus leukemic progression. Orimo et al. have reported that stromal fibroblasts present in invasive human breast cancer promote tumor growth and angiogenesis through elevated SDF/CXCL12 secretion [7]. Together, these reports advocate the importance of complex interactions between CLL and other tumor cells with their microenvironment for increased proliferation leading to disease progression. In B cells, such interactions involve cytoskeletal changes possibly mediated by stromal microenvironment leading to enhanced B cell activation by BCR clustering [8,9]. Similar mechanisms may impose altered cytoskeletal changes in CLL cells leading to better survival and proliferation. Therefore, identification of molecular network involved in modification of LN microenvironment by CLL cells will lead to a better understanding of the disease. In an attempt to understand the molecular basis of stromal associated regulation of CLL progression and its prognostic implication we performed a gene expression profiling (GEP) of CLL cells from PB, BM and LN. We also performed transcriptome analysis of PB-CLL cells from patients with good versus poor prognosis to identify stromal gene signatures associated with disease aggressiveness. We identified two genes, MYLK and CAV2, whose transcript and protein expression is upregulated in patients with poor prognosis than good prognosis and significantly associated with patient's outcome.

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma (NHL) which is one of the most aggressive lymphomas. Despite recent improvements in therapies, the development of therapy-resistance is still a major problem; therefore, in order to understand the molecular basis of therapy-resistance, stable therapy-resistant MCL cell lines have been established by us. Based on the gene expression profiles of these cell lines, Polo-like kinase 1 (PLK1) was chosen as a therapeutic target. In this paper, we demonstrate a significant antilymphoma effect of targeting PLK1 in therapy-resistant MCL cells and primary MCL cells from refractory patients. PLK1 knockdown with the antisense oligonucleotide (ASO)/or small molecule inhibitor BI2536 showed significantly decreased proliferation and increased apoptosis in therapy-resistant MCL cell lines and MCL primary cells. Additionally, the direct proteinprotein interaction partners of PLK1 were mapped using ingenuity pathway and confirmed the level of association of these partners with PLK1 based on their expression changes following PLK1 knockdown using real-time PCR. Results suggest that PLK1 is a viable target for the treatment of therapy-resistant MCL.

1781 Chronic Lymphocytic Leukemia (CLL), the most prevalent adult B-cell malignancy in western countries, is a highly heterogeneous with a very variable clinical outcome. Emerging evidence indicates that the stromal tumor microenvironment (STME) play important roles in the pathogenesis of CLL. However, the precise mechanism and molecules of STME involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from 53 patients’ lymph node (LN) (n=15), bone marrow (BM) (n =18), and peripheral blood (PB) (n=20). Using significant analyses of microarray (SAM), gene set enrichment analyses (GSEA), and ingenuity pathway analyses (IPA), among the major pathways associated with the differentially expressed genes, a cytoskeleton genes associated with stromal signatures are the focus of this report. Of these molecules, a significant number of molecules including: LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were overexpressed and ITGB2, DLC1 and ITGA6 are under expressed in LN-CLL compared to BM-CLL and PB-CLL indicating a role of LN-mediated TME in CLL cell survival/progression. Among these genes, expression of myosin light chain kinase (MYLK), caveolin 1 (CAV1) and caveolin 2 (CAV2) correlated with clinical outcome (see adjacent Figure) as determined by time to treatment. We recently reported the role of a CAV1 in LN microenvironment-induced immune tolerance in CLL and possibility of their involvement in CLL cytoskeleton (Gilling et al, 2012). In the present study we report aberrant expression of other cytoskeleton genes such as MYLK and CAV2 are involved in the regulation of CLL cell survival in the stromal microenvironment affecting other members of the cytoskeletal signature via actin cytoskeleton signaling, integrin signaling and Pak signaling. In addition, MYLK and CAV2 are also involved in regulation of CLL proliferation. Together our studies show that members of the stromal signature particularly in the CLL cells from lymph nodes regulate the CLL cell survival and proliferation and thus leukemic progression. Figure: Association of MYLK expression with time to first treatment Figure:. Association of MYLK expression with time to first treatment Disclosures: No relevant conflicts of interest to declare.