ALBERT

Description: Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.

Description: Abstract 287 Mantle cell lymphoma (MCL) is a lymphoproliferative disorder of mature B-cells with an aggressive course and short survival. The proteasome inhibitor bortezomib (BZM) induces clinical responses in up to 50% of patients. Conversely, in half of the cases the lymphoma cells are intrinsically resistant or rapidly develop resistance to BZM. To investigate the mechanisms of BZM resistance, we generated HBL2 and JEKO bortezomib resistant (HBL2-BR, JEKO-BR) derivative lines by continuous culture in sub-lethal concentrations of BZM. After several months, clones of HBL2-BR and JEKO-BR were obtained showing BZM IC50 at 48h of 41.6 and 44.6 nM, compared to 6 and 4.9 nM for the respective parental lines. Acquired resistance to BZM remained stable over months but gradually decreased with extended passages in the absence of BZM, suggesting adaptive changes rather than a single gene mutation as the basis of BZM resistance. BR cells exhibited higher proteasome activity, which was dose-dependently inhibited by higher concentrations of BZM. However, BR cells were able to survive with lower proteasome activity than the parental cells, indicating that BR cells had acquired additional changes. To investigate these changes, we use gene expression profiling (GEP) on Affymetrix U133A plus 2 arrays to compared HBL2-BR (in triplicate) and JEKO-BR (in duplicate) subclones to the corresponding parental lines. Unexpectedly, Gene Set Enrichment Analysis (GSEA) of microarray data revealed reduced expression of the mature B-cell gene signature (including genes for CD19, BLNK, SPIB, SYK) and increased expression of plasma cell differentiation signatures (including genes for CD38, IRF4, BLIMP, CD138) in both HBL-2 BR and JEKO-BR. BR lines also expressed higher protein levels of the master plasma cell regulators BLIMP and IRF4, but did not show enhanced expression of the secretory program controlled by XBP1. Flow cytometry analysis confirmed that BR cells had dramatically reduced expression of B-cell surface markers, including CD19, CD24 and CD52, and expressed plasma cell markers, such as CD38 and CD138. Consistent with a partial plasmacytoid phenotype, BR cells tended to be somewhat larger and more granular than parental cells. Loss of BZM resistance over months of culture in the absence of BZM was paralleled by the recovery of CD19 and CD24 expression and down-regulation of CD38, supporting a mechanistic link between the acquisition of a plasmacytoid phenotype and BZM resistance. We have previously shown that the MCL cell lines Mino and REC-1 are less sensitive to BZM than HBL-2, JEKO and most other MCL cell lines. Here we found that these constitutively resistant cells also showed plasmacytoid features including CD38 and CD138 surface expression, increased granularity and size, and an enlarged endoplasmic reticulum (ER). Combined these changes may enhance the ability of the cells to deal with an increased protein load due to bortezomib inhibition. In addition, we also observed higher expression of IRF4 and its target genes in the constitutively resistant cells, as well as higher IRF4 and CD38 expression in primary tumor cells of patients with poor response to BZM. Given the important role of IRF4 as a survival factor in multiple myeloma, we tested whether BZM treatment could decrease IRF4 expression in MCL cells. Indeed, within 24 hours BZM dose-dependently decreased IRF4 expression and the degree of downregulation of IRF4 correlated with the induction of apoptosis. Knockdown of IRF4 expression by shRNA has been shown to be toxic to myeloma cells (Shaffer et al, Nature 2008). Surprisingly, we found a similar toxic effect of IRF4 knockdown using the same inducible shRNA system in the MCL cell lines HBL2, JEKO and REC, which was more prominent in the latter BZM resistant cell line. These results identify loss of IRF4 expression as an additional mechanism by which BZM may induce cell death. However, overexpression of IRF4 in MCL cells is not sufficient to induce bortezomib resistance, indicating that several components of the plasma cell program cooperate to protect cells from BZM induced apoptosis. Furthermore, we have identified markers of BZM resistance that may be clinically relevant predictors of outcome. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 412 Chronic Lymphocytic leukemia (CLL), the most common leukemia in Western countries, is a clinically heterogeneous disease characterized by the accumulation of mature B lymphocytes. Disease progression is driven by cell survival and proliferation signals provided by the tumor microenvironment through different receptors including the B-cell receptor, TNF, chemokine and cytokine receptors, and through other cell-cell interactions. ON 01910.Na (Onconova, Therapeutics) belongs to the family of styryl benzyl sulfones, a novel family of non-ATP competitive compounds that are currently under development as potential anticancer treatments. These compounds have shown activity in a variety of xenograft mouse models. Interestingly, it has recently been described that ON 01910.Na exerts potent antitumor activity against Mantle Cell Lymphoma (MCL) cells by inhibition of PI-3K/Akt/mTOR pathway and downregulation of Cyclin D1 translation. Here, we evaluated the cytotoxic effect of ON 01910.Na after 48 hours of in vitro exposure on PBMCs from CLL and normal donors by flow cytometry using AnnexinV-PE. We co-stained with CD3-APC and CD19-FITC to assess the effect on B and T cells separately. ON 01910.Na induced apoptosis of the leukemic cells in all CLL samples tested (n=24), without affecting T-cell viability. Notably, ON 01910.Na did not affect B and T cells from normal donors at concentrations that induced apoptosis of CLL cells. Thirteen CLL samples were highly sensitive towards ON 01910.Na (mean B-cell IC50 0.71 μM) a concentration readily achieved in phase I clinical trials, eight samples were sensitive (mean B-cell IC50 1.38 μM) and three showed moderate sensitivity (mean B-cell IC50 4.26 μM). In contrast, the IC50 of T-cells was 〉8 μM in all samples. No significant differences were found in ON 01910.Na cytotoxicity against CLL expressing mutated (n=9, mean B-cell IC50 1.45 μM) or unmutated (n=15, mean B-cell IC50 1.34μM) IgVH sequences. ON 01910.Na was also effective against tumor cells having 17p (n=4, mean B-cell IC50 1.45 μM) or 11q deletions (n=3, mean B-cell IC50 0.96 μM). ON 01910.Na activated the mitochondrial apoptotic pathway, as shown by flow cytometry using conformation specific antibodies to detect activation of Bax and Bak, leading to mitochondrial depolarization (measured by mitotracker) and caspase-3 activation. ON 01910.Na also induced the upregulation of the proapoptotic BH3-only proteins Noxa and Bim and decreased expression of Mcl-1 at 4 hours, well before the onset of apoptosis. Inhibition of Noxa expression by retroviral transfection reduced the sensitivity to drug by 60%. ON 01910.Na induced reactive oxygen species (ROS) that may contribute to Noxa induction. Consistently ROS blockade using N-acetyl-cysteine reduced ON 01910.Na cytotoxic activity. To further delineate the biological processes underlying ON 01910.Na induced apoptosis, we performed Gene Expression Profiling (GEP) in CLL cells treated in vitro for 4 and 10 hours with ON 01910.Na. GEP revealed the induction of Noxa and ATF3, consistent with an oxidative stress response. Noteworthy, Gene Set Enrichment Analysis (GSEA) of microarray data showed an induction of an AP-1 gene signature, (False Discovery Rate (FDR) = 0.10), validated by the nuclear accumulation of c-jun at 4 hours and a FOXO gene signature (FDR=0.13) that correlated with the upregulation of the proapoptotic BH3-only protein Bim. Strikingly, ON 01910.Na repressed B-cell receptor (FDR=0.023), NF-kB (FDR=0.084) and PI3K gene signatures. Consistent with inhibition of the BCR/PI3K/AKT axis, ON 01910.Na inhibited AKT phosphorylation by 65% after in vitro BCR activation of CLL cells. Our results identify ON 01910.Na as a promising agent in the treatment of CLL with an interesting dual mechanism of action: activation of apoptotic stress signals leading to Noxa and BIM up-regulation, combined with inhibition of the BCR/PI3K/AKT pathway that can block microenvironment-induced survival and proliferation signals. These data support the development of ON 01910.Na in CLL and a clinical trial has been initiated at our institution. Disclosures: No relevant conflicts of interest to declare.

Description: ROR-1, an orphan receptor tyrosine kinase (RTK) carrying an extracellular WNT binding motif, is highly expressed in many tissues during development. Expression of ROR-1 in lymphoid cells has first been noted in a gene expression profiling study of B-cell chronic lymphocytic leukemia (B-CLL), where ROR-1 was expressed in CLL cells but not in diffuse large B-cell lymphoma or in normal B-cells (Rosenwald, 2001). B-CLL is the most common type of leukemia in western countries and is characterized by an accumulation of mature B lymphocytes in the blood and lymphoid tissues. The prolonged survival of the malignant cells in-vivo is dependent on signals derived from the microenvironment. ROR-1 recently has been implicated in mediating stroma dependent survival signals, with WNT5a being a putative ligand for ROR-1 (Fukuda, 2008). In addition, an RNAi screen identified ROR-1 as a tyrosine kinase with anti-apoptotic effects in Hela cells (MacKeigan, 2005). With this background, we have further examined the role of ROR-1 in the survival of B-cell malignancies. We first characterized ROR-1 surface expression by flow cytometry on various lymphoid cell lines and primary cells. We found ROR-1 was highly expressed in mantle cell lymphoma (MCL) cell lines and moderately expressed in the CLL derived EBV positive cell line MEC-1. In contrast, cell lines derived from other B-cell (BJAB, SUDHL-4) and T-cell (Jurkat) malignancies had no ROR-1 expression. Moreover, ROR-1 was highly expressed in primary MCL cells (n= 5) and in follicular lymphoma (n=1). Comparable levels of expression were also detected in CLL cells independent of IgVH mutation status (IgVH unmutated: n=11; IgVH mutated: n= 8). To investigate whether and how ROR-1 could activate intracellular signaling pathways, we chose a monoclonal antibody to induce ROR-1 activation through receptor dimerization. In response to antibody binding, ROR-1 was tyrosine phosphorylated within minutes in a dose dependent manner in MCL cell lines and in primary CLL and MCL cells. Intriguingly, the MCL cell line UPN-1, bearing high ROR-1 surface expression (specific MFI ratio of 19), showed constitutive ROR-1 phosphorylation that further increased after crosslinking. Several studies on ROR-2, the closest ROR-1 homolog, reported activation of the non-canonical WNT signaling pathway and c-Jun N-terminal kinase (JNK) activation. We therefore investigated whether ROR-1 cross-linking could activate JNK and/or lead to stabilization of beta-catenin, the main mediator of canonical WNT signaling. Indeed, we detected JNK phosphorylation in MCL cell lines and in primary CLL cells, while we could not detect beta-catenin activation. In conclusion, ROR-1 surface expression is high in MCL cell lines and primary cells from CLL, MCL and FL patients. ROR-1 crosslinking leads to ROR-1 phosphorylation in both cell lines and primary cells followed by activation of the non-canonical WNT/JNK pathway. Our findings suggest that ROR-1, which plays important roles in embryonic development and organogenesis, could play an important role in the pathogenesis of low grade lymphomas and B-CLL.