Introduction: BTK is involved in B-cell receptor (BCR) signal transduction and is an established target for the treatment of chronic lymphocytic leukemia (CLL) (Byrd, NEJM, 2013). ACP-196 is a novel, ...potent second generation BTK inhibitor, which binds covalently to Cys481 in the ATP-binding pocket of BTK. IC50determinations on nine kinases with a cysteine in the same position as BTK showed ACP-196 to be more selective than the first-in-class BTK inhibitor, ibrutinib (Covey, AACR, 2015). We present data evaluating the anti-tumor effects of ACP-196 in established murine models of CLL.
Methods: Two distinct murine models were used for these studies. In the TCL1 adoptive transfer model, leukemic cells from Eμ-TCL1 transgenic mice were transplanted into C57BL/6 mice, resulting in a CD5+/CD19+ leukemia with peripheral blood, spleen and nodal involvement. ACP-196 treatment in drinking water (0.16 mg/mL) commenced when recipient mice had > 10% CD5+/CD19+ leukocytes in the peripheral blood. Mice were followed for survival. Separate cohorts were sacrificed for pharmacodynamic analyses after 1 and 4 weeks of treatment. In the second model, NSG mice received primary human CLL cells. The xenografted human CLL cells have comparable tumor biology (including active BCR signaling) to activated human lymph node resident CLL cells (Herman, Leukemia, 2013). PBMCs harvested from CLL patients were adoptively transferred at 1 x 108 cells per mouse. ACP-196 was initiated on day -1 (at the time of busulfan priming) at multiple doses ranging from 0.006 to 0.3 mg/mL in drinking water.
Results: In the TCL1 model, treatment with ACP-196 showed > 90% BTK occupancy of BTK after 1 and 4 weeks of therapy. ACP-196 inhibited BCR signaling as shown by decreased autophosphorylation of BTK and reduction in surface expression of the BCR activation markers CD86 and CD69. After 1 week of ACP-196 inhibited BCR signaling as shown by a 6-fold reduction of autophosphorylation of BTK in the presence of anti-IgM, and surface expression of the BCR activation markers CD69 and CD86 were decreased by 47% and 57% respectively. Inhibition of BTK and downstream BCR activation was maintained through at least day 28 of treatment. Most notably, ACP-196 treatment resulted in a significant increase in survival compared with mice receiving vehicle (median 81 vs 59 days, respectively; P =0.02). In the NSG xenograft model, ACP-196 at the times examined did not cause a significant treatment-induced lymphocytosis in the patients evaluated (n=6). After 4 weeks of treatment with ACP-196, the NSG mice were sacrificed, and BCR signaling activity and tumor burden in the spleen were evaluated. ACP-196 treatment showed decreases in phosphorylation of PLCγ2 and ERK (P <0.02) as expected with BTK inhibition. Additionally, a significant reduction was observed in the percentage of proliferating cells in mice treated with ACP-196 compared to vehicle control (as determined by Ki67 staining; median 18% (Intraquartile Range (IQR): 13-38) and 6% (IQR: 2-15), respectively, P =0.02). Lastly, a significant reduction in total tumor burden in the spleen was observed in the mice treated with ACP-196 (median reduction of 33%) compared with the vehicle treated mice (P =0.04).
Conclusions: ACP-196 is a potent inhibitor of BTK as measured by inhibition of BCR activity, reduced tumor proliferation and increased survival. Overall, ACP-196 showed statistically significant efficacy in two murine models of CLL and is currently in Phase 3 trials for treatment-naive (ClinicalTrials.gov NCT0247568) and previously treated high-risk CLL (ClinicalTrials.gov NCT02477696).
This work was supported by the Intramural Research Program of NHLBI, NIH, R01CA197870, K23 CA178183-02, and Acerta Pharma.
Gulrajani:Acerta Pharma: Employment. Krantz:Acerta Pharma: Employment. Covey:Acerta Pharma BV: Employment, Equity Ownership, Patents & Royalties. Lannutti:Acerta Pharma: Employment. Izumi:Acerta Pharma: Employment, Equity Ownership, Patents & Royalties. Ulrich:Acerta Pharma: Employment. Byrd:Acerta Pharma BV: Research Funding. Wiestner:Pharmacyclics: Research Funding. Johnson:Acerta Pharma: Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 3775
Olfactomedin 4 (OLFM4) was initially identified as a gene highly induced in myeloid stem cells by G-CSF treatment and independently as a gene highly expressed in colon cancers. OLFM4 ...was predicted in a bioinformatics analysis as associated with neutrophil specific granules. We analyzed the expression of OLFM4 mRNA in myeloid cells from normal human bone marrow and demonstrated that expression of OLFM4 mRNA is similar to the expression of LCN2 which codes for the specific granule protein NGAL (Figure 1), but distinct from expression of mRNA for myeloperoxidase and gelatinase which are marker proteins for azurophil granules and gelatinase granules, respectively. Subcellular fractionation of peripheral blood neutrophils demonstrated complete co-localization of OLFM4 with NGAL, and stimulation of neutrophils with fMLP or PMA resulted in co-release of NGAL and OLFM4, indirectly proving that OLFM4 is a genuine constituent of neutrophil specific granules.
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Interestingly, immunohistochemistry showed OLFM4 expression in only a subset of neutrophils (figure 2). We suspected that this might be dependent on the antibody, but two different commercial antibodies and an in-house antibody raised against a synthetic OLFM4 derived peptide, all polyclonal, showed similar patterns. Flow cytometry confirmed the existence of two populations of neutrophils, one expressing OLFM4 the other not.
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Immunohistochemistry of bone marrow cells showed that OLFM4 appears in myelocytes and is maintained in the cells during further maturation of the cells to segmented neutrophils. Again, only 30% of the neutrophil precursors from bone marrow stain positive for OLFM4 indicating, that different subsets of human neutrophils may exist.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mantle Cell Lymphoma (MCL) is a malignancy of mature B-cells. MCL has a poor prognosis and a limited response to traditional chemotherapy. Bortezomib (BZM), a new powerful inhibitor of the ...proteasome, can induce responses in up to 50% of relapsed MCL patients, suggesting that in at least half of the patients the lymphoma cells are intrinsically resistant to BZM or rapidly develop resistance during single agent therapy. To investigate possible mechanisms of BZM resistance, we cultured MCL cell lines continuously in sub-lethal concentrations of BZM that were then gradually increased. Resistance was slow to develop taking several months for truly resistant clones to grow out. We generated a bortezomib resistant (BR) clone of HBL-2 with an IC50 of 30nM compared to 5nM in the parental clone and several BR subclones of Jeko-1, the most resistant of which had an IC50 of 200nM compared to 3nM for the parental clone. All BR subclones also showed decreased sensitivity to three other proteasome inhibitors: MG-132, Lactacystin, and NLVS. The increase in IC50 to these drugs was between 3 and 8-fold, consistent with more off-target effects of these drugs compared to BZM. BAY11-7082, an inhibitor of NF-kB signaling, maintained its activity against the resistant cells. Resistance to BZM, once acquired, has remained stable over several months. This is remarkable because the resistant subclones grow significantly slower than the parental lines, even after having been removed from selection for extended periods of time. Consistent with slower cell proliferation, we found reduced Cyclin D1 protein expression in the BZM resistant Jeko clones; however, mRNA levels were comparable to the parental line, indicating that changes in Cyclin D1 protein translation and/or stability may be responsible for the decreased proliferation. BZM resistance has been associated with up-regulation of proteasome components and heat-shock proteins. Indeed, in the resistant HBL-2 subclone we found marked upregulation of two proteasome components (PSMA5 and PSMC1) and of Hsp70 by RT-PCR, but there was only a small change in Hsp70 protein expression. Nevertheless, upregulation of these genes could be part a more global gene expression response as seen with ER-stress and could thus reflect an adaptive change to BZM in the HBL-2 BR subclone. All three Jeko BR clones in contradistinction showed only minor changes in PSMA5, PSMC1 and Hsp70 mRNA expression and surprisingly had markedly reduced Hsp70 protein levels. Thus, in these subclones, BR resistance appears to correlate primarily with changes affecting cell cycle control. We conclude that resistance to BZM may be determined by several mechanisms that affect cell cycle control as well as expression of proteasome components and heat-shock proteins. While the slow development of resistance suggests adaptive changes, its persistence over time is more consistent with mutations or other genomic alterations that are not readily reversible. Ongoing studies aim to more precisely define the basis for BZM resistance in MCL.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mantle cell lymphoma (MCL), characterized by a t(11;14) translocation that results in up-regulation of cyclin D1, is incurable with standard chemotherapy. Recent phase II studies have shown that ...bortezomib (BZM), an inhibitor of the proteasome, can induce responses in about 50% of pre-treated MCL patients. However, the molecular mechanisms that mediate either chemosensitivity or resistance to BZM in MCL remain largely unknown. In this study, we used a panel of MCL cell lines to investigate molecular mechanisms of response to BZM. In 11 MCL cell lines we found a bimodal pattern of chemosensitivity to BZM; the resistant group, REC-1, Mino, and NCEB-1, had an IC50>10nM (median 12.9 nM), while the sensitive group, Granta-519, JVM-2, Jeko-1, HBL-2, UPN1, SP-53, SP-49, and Z-138, had an IC50<10nM (median 5.9nM). No correlation between BZM resistance and p53 mutations was apparent, arguing against a role for this common chemotherapy resistance mechanism. To test whether the differences in sensitivity to BZM might be mediated by drug export mechanisms we measured P-gp activity using the rhodamine efflux assay. We found that more than 50% of the REC-1 (resistant), SP-53, and SP-49 cells (both sensitive) excluded the rhodamine dye, while all other cell lines showed only minimal or no activity, arguing against a role of P-gp in BZM resistance. To determine whether high proteasome activity or reduced sensitivity of the proteasome to inhibition could cause resistance to BZM we quantified proteasome activity by measuring the cleavage of the labeled substrate LLVY-AMC. Basal proteasome activity was comparable except in the sensitive cell line JVM-2, which had an activity 71% higher than the second highest; dose dependent inhibition was similar in all cell lines. BZM can interfere with components of the NFkB pathway, and this effect has been proposed to mediate cytotoxicity in MCL. We measured the relative activation of the NFkB pathway by quantifying p50, p52, p65, c-Rel and Rel-B nuclear factors with an ELISA assay. Cell lines with high and low expressions of NFkB nuclear factors were equally sensitive, arguing against a major role for this pathway in determining sensitivity to BZM. Heat shock proteins (Hsp) have been reported to confer resistance to BZM in lymphoid cell lines. However, in our hands, Hsp27, Hsp70 and Hsp90 were equally expressed between resistant and sensitive cells, and there was no consistent pattern of regulation of these proteins in response to BZM. Recently, NOXA has been reported to mediate BZM induced apoptosis. Indeed, all cell lines showed up-regulation of protein levels of NOXA when exposed to BZM in excess of their IC50 concentrations, suggesting that determinants of resistance are upstream of NOXA. Given the cardinal role of cyclin D1 in MCL, we hypothesized that effects on cell cycle control could be responsible for chemosensitivity. Upon BZM exposure sensitive cells were arrested in G2/M, whereas resistant cell lines either accumulated in G1 (Mino) or, at higher concentrations, underwent apoptosis without arresting in any specific phase. We conclude that BZM can overcome conventional mechanisms of drug resistance and that an effect on cell cycle control may determine BZM activity in MCL.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mantle cell lymphoma (MCL) is an aggressive and incurable B-cell lymphoma for which new treatment options are needed. Recent phase II clinical trials reported response to the proteasome inhibitor ...bortezomib (BZM) in up to 50% of pre-treated patients. Despite the successful use of BZM in the clinic, the precise molecular mechanisms underlying sensitivity or resistance to BZM in MCL remain largely unknown. To address this issue, we used U133A 2.0 microarrays to analyze gene expression in MCL cells from peripheral blood of 5 patients with previously untreated leukemic MCL. Samples were collected immediately before (0h) and at 3, 6, 24, and 72 hours after administration of BZM (1.5 mg/m2). After the blood collection at 72 hours, a second dose of BZM was given, and cells were collected 24 hours later. Two patients had major reductions in peripheral ALC already at 24h from dose 2 and normalized their blood counts by day 21 (sensitive), 1 patient had no change over a full course of 4 injections (resistant), and 2 patients had some decrease in ALC (intermediate). Genes differentially expressed with treatment were ranked according to the degree of correlation with time (Pearson). We used gene set enrichment analysis (GSEA) to detect distinct functional gene expression signatures; the most consistently up-regulated of which was a signature composed by proteasome and chaperone genes. To confirm and expand these findings, we exposed 10 MCL cell lines (7 sensitive, IC50<10nM; 3 resistant IC50>10nM) to 10nM of BZM and analyzed gene expression at 1, 3, 6 and 24 hours. The proteasome signature was again dominant, and the majority of the up-regulated genes in both clinical and cell line samples shared binding motifs for the NRF, MAF, ATF and HSF families of transcription factors (TF). Thus genes up-regulated by BZM in vivo and in cell lines predominantly belonged to a functional response to oxidative and/or endoplasmic reticulum (ER) stress. Under physiologic conditions, this is thought to help restore homeostasis and protect from apoptosis. This response could therefore contribute to drug resistance or be a marker of an overwhelming insult before the cells undergo apoptosis. To address this issue, we investigated differences in response to BZM between sensitive and resistant cell lines. The proteasome signature was more strongly up-regulated in sensitive cells than in resistant cells, and the ER-stress response as measured by genes controlled by the NRF and MAF family of TFs was also more highly expressed in the sensitive group. Consistently, expression of HMOX1, which encodes a key enzyme in the antioxidant response, was increased by 32× at 24h in the sensitive group, but only by 4× in the resistant group; the expression of DDIT3, a transcription factor implicated in a pro-apoptotic response to ER-stress was 5.5-fold up-regulated in the sensitive cells but only 1.4-fold in the resistant cells. We conclude that in sensitive cells BZM induces an overwhelming ER-stress response with high expression of proteasome components and chaperone proteins that could serve as a predictor of response to BZM.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Bortezomib, a potent inhibitor of the 26S proteasome, has remarkable activity against some lymphoid malignancies, particularly multiple myeloma and mantle cell lymphoma (MCL). The observed 30–50% ...clinical response rate to bortezomib as a single-agent in relapsed MCL is believed to be mediated mainly by inhibition of the NFkB signaling pathway. Recently, Hsp27 has been shown to confer resistance to bortezomib in a lymphoid cell line. However, much less is known about the mechanisms underlying anti-tumor activity of or resistance to bortezomib in MCL. To address these questions we studied 10 MCL cell lines with t(11;14)(q13;q32) as in vitro models. IC50 values were measured by the MTT cytotoxicity assay for both bortezomib and the BAY 11-7082 compound, a specific inhibitor of the NFkB pathway. The cell lines showed different profiles of response to bortezomib and were grouped according to their IC50 values as sensitive (S) (Granta 519, Jeko-1, SP-49, UPN-1), intermediate (In) (HBL-2, JVM-2, Z-138) and resistant (R) (Mino, NCEB-1, SP-53). The mean IC50 for bortezomib in the R group was 3 times higher than the mean IC50 of the S group. The cell lines also showed very different profiles of response to the NFkB-specific inhibitor BAY 11-7082, but grouped differently, suggesting that other mechanisms, in addition to the NFkB pathway, participate in the anti-neoplastic effect of proteasome inhibition in MCL. Resistance to both drugs had no correlation with P-glycoprotein activity as measured by the rodhamine efflux assay. We performed gene expression profiling on Affymetrix U133A 2.0 arrays of all 10 cell lines. Using GeneSpring software (Agilent), we normalized expression to the mean of the S group and identified 79 transcripts that showed a dose response behavior; that is, genes whose mean expression was 1.5x higher in the In versus the S group and 1.5x higher in the R versus the In group. Conversely, 55 transcripts followed the opposite trend, being down-regulated at least 1.5x in both comparisons. This list contained genes important in apoptosis control and in transmembrane transport, but was most notable for an overexpression of stress response genes in the R group. Notably, expression of Hsp70 was almost 10-fold higher in the R versus S group. We therefore analyzed expression of the HSP70 as well as HSP27 and HSP90 proteins by Western blotting. Hsp70 protein was highly expressed in all MCL cell lines and there was no discernable correlation with bortezomib resistance for any of the HSP-proteins analyzed. Other resistance mechanisms appear more important in MCL and we are currently testing further candidate genes for their ability to confer resistance to bortezomib in MCL.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Bortezomib is more active against mantle cell lymphoma (MCL) than against most other lymphoma subtypes. Nevertheless, up to half of patients with MCL have bortezomib resistant disease. Factors ...contributing to intrinsic resistance to bortezomib have not been determined. Here we used a panel of eight bortezomib sensitive (median IC50 5.9 nM) and three relatively bortezomib resistant cell lines (median IC50 12.9 nM) to investigate differences in tumor biology that could determine sensitivity to bortezomib. Bortezomib effectively inhibited high baseline proteasome activity and induced a comparable degree of proteasome inhibition in both sensitive and resistant cells. At 10 nM, bortezomib induced the proapoptotic BH3-only protein Noxa in sensitive but not resistant cells. At higher concentrations of bortezomib, however, Noxa was also upregulated in resistant cells and this effect was sufficient to induce apoptosis. Silencing of Noxa with siRNA rescued these cells from apoptosis, arguing against a defect in Noxa regulation or function as the basis of bortezomib resistance. Bortezomib was equally effective against cells with high and low constitutive NF-κB signaling. Also, sensitive and resistant MCL cell lines showed comparable activation of the AKT pathway. We conclude that bortezomib can overcome classic mechanisms of resistance to apoptosis and that determinants of bortezomib sensitivity in MCL are due to differences in signaling or stress pathways upstream of Noxa.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
