The Role BAFF and BAFF Receptors in Macrophage Induced Drug Resistance of Multiple Myeloma

Abstract Background: B-cell–activating factor (BAFF) is a member of TNF family that critical for maintenance of B-cell development and homeostasis. BAFF also modulates the proliferation, survival and drug resistance of multiple meyloma (MM) cells. BAFF can be secreted by neutrophils, monocytes,dentritic cells and macrophages. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by a cell-to-cell interaction between MM cells and macrophages. We supposed that the interaction of BAFF and BAFF receptors plays a role in macrophages induced MM resistance. Methods: First, we detected the expression levels of BAFF and its three receptors in MM cells were detected by semiquantitative real time-polymerase chain reaction (qPCR) and flow-cytometry. The concentration of BAFF in the culture supernatants of MM cell lines was detected by ELISA. Second, we collect peripheral blood monocytes form healthy donors. Monocytes were induced into macrophages by culturing with M-CSF for 7 days.BAFF expression level in macrophages was detected by qPCR, flow-cytometry and ELISA. Then MM cells were sole-cultured or coculture with macrophages for the indicated time (usually 24 h), after that bortezomib was added to the culture system. Cell viability and apoptosis of MM cells were verified by MTT and flow cytometry.At last,the recombinant human BAFF were added to MM cells, MTT and flow cytometry were used for detection of cell viability and apoptosis of MM cells. Results: Two receptors of BAFF, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA), are highly expressed in various MM cell lines evidenced by real-time PCR and flow cytometry. The expression of BAFF in PBMC-induced macrophages are heterogeneous. We verify macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, 8226 and OPM2) with PBMC-derived macrophages from healthy donors. Functional studies show that recombinant human BAFF rescues RPMI8226 myeloma cells from dexamethasone-induced apoptosis. Conclusions: Our data showed that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors. Further study will focused on the mechanism of interaction between BAFF and BAFF receptors. Disclosures No relevant conflicts of interest to declare.

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Interactions Between BAFF and BAFF Receptors Are Involved in Macrophage-Induced Bortezomib Resistance in Myeloma

Blood ◽

10.1182/blood.v128.22.4429.4429 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4429-4429

Author(s):

Jing Chen ◽

Donghua He ◽

Xing Guo ◽

Qingxiao Chen ◽

Xuanru Lin ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Resistance ◽

Western Blot ◽

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Counting ◽

Therapeutic Modality ◽

Second Primary ◽

Induced Apoptosis

Abstract Background:B-cell-activating factor (BAFF) is a member of the TNF family that critical for maintenance of B-cell development and homeostasis. BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI) are three BAFF receptors. It has been reported that BAFF is expressed by neutrophils, monocytes, dentritic cells and macrophages and modulates the proliferation, survival and drug resistance of multiple myeloma (MM) cells. Our previous study showed that, macrophages protect MM cells from drug-induced apoptosis by direct interaction with MM cells. We hypothesized that BAFF/BAFF receptors play a role in macrophage-induced bortezomib resistance in myeloma. Methods: First, the expression levels of BAFF and its three receptors in primary MM cells, MM cell lines and peripheral blood monocyte(PBMC)-induced macrophages were detected by semiquantitative real time-polymerase chain reaction (qPCR),Western blot and flow-cytometry. Also the concentration of BAFF in the supernatants of MM patients' bone marrow, MM cell lines and macrophages were determined by ELISA. Second, Primary MM cells and MM cell lines were cocultured with macrophages for the indicated time (usually 4-6h and 24h), for some experiments, we added bortezomib to the coculture system. Cell viability and apoptosis of MM cells were verified by Cell Counting Kit-8(CCK8) after treated with recombinant human (rh) BAFF, BAFF neutralizing antibody and BAFF siRNA. The interactions between BAFF and its receptors are unveiled by flow-cytometry. Then, cell survival signaling activations that may confer MM drug resistance were examined by Western blot. Results: Two receptors of BAFF, TACI and BCMA were highly expressed in various MM cell lines. The expressions of BAFF in PBMC-induced macrophages were heterogeneous. Functional studies showed that rhBAFF promoted RPMI8226 and ARP1 myeloma cells growth (P<0.05) and protected them from bortezomib-induced apoptosis (P<0.05). Then we verified macrophage-mediated MM drug resistance by directly coculturing MM cells (ARP-1, RPMI8226) with PBMC-derived macrophages from healthy donors. The macrophage-induced bortezomib resistance was attenuated by neutralizing antibodies(P<0.05) and siRNA of BAFF(P<0.01) . Next we found that in MM cells cocultured with macrophages, bortezomib-induced PARP and caspase-3 cleavages were highly repressed and phosphorylated Src ,AKT and Erk1/2 were upregulated which indicated that BAFF-mediated MM drug resistance may be through ERK1/2 and Src pathway .In addition, BAFF induced activation of NF-κB2,a pathway critical for the growth and survival of these cells. Conclusions: Our data show that macrophage might induce drug resistance of MM cells by the interaction of BAFF and BAFF receptors, leading to a reduction in caspase proteins and subsequent activation of Src and Erk1/2 kinases and NF-κB2 pathways .These studies reveal a promising unknown role for BAFF/BAFF receptors, suggesting that targeting macrophage-MM interactions may represent a promising therapeutic modality. Disclosures No relevant conflicts of interest to declare.

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Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽

10.1182/blood.v114.22.1737.1737 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1737-1737

Author(s):

David M. Lucas ◽

Ellen J. Sass ◽

Ryan B. Edwards ◽

Li Pan ◽

Gerard Lozanski ◽

...

Keyword(s):

Drug Resistance ◽

T Cells ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Multi Drug Resistance ◽

Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

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5-Aza-2-Deoxycytidine Regulates Wnt Beta-Catenin Pathway in B Cell Lymphoma

Blood ◽

10.1182/blood.v126.23.4810.4810 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4810-4810

Author(s):

Xinyu Li ◽

Lingyu Geng ◽

Xiangxiang Zhou ◽

Kang Lu ◽

Peipei Li ◽

...

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Cell Lines ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Beta Catenin ◽

Cell Functions ◽

Protein Expressions

Abstract Introduction: The Wnt/beta-catenin pathway is aberrantly activated in B cell lymphomas, unphosphorylated beta-catenin accumulates and translocates into the nucleus, regulates the expression of c-myc, cyclinD1 and many other target genes which govern fundamental cell functions, such as proliferation, cell cycle regulation and apoptosis. Methylation is a highlight of epigenetic regulation research which also occurred in lymphoma, but the concrete mechanism of how the demethylation drug 5-aza-2-deoxycytidine affect Wnt/beta-catenin pathway is still unknown. This study was designed to illuminate the implications on Wnt/beta-catenin pathway via demethylation 5-aza in B cell lymphoma. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from samples of 30 primary CLL patients. The PBMCs contained more than 90% of CD19+ B lymphocytes, which were detected by flow cytometry and were referred to as primary CLL cells. The activation of Wnt/beta-catenin pathway and DNMT-1 of B cell lymphoma cells lines (MEC-1, LY8, Jeko-1, Grant519, mino and sp53) and the 30 patients were detected by qPCR and western blot. The expressions of beta-catenin in 20 cases of B cell lymphoma tissues were measured by IHC. The B cell lines and PBMCs from 10 primary CLL patients were given 5-aza-2-deoxycytidine in different concentrations, the effects in the pathway and apoptosis were observed by WB and flow cytometry. Results: The expressions of beta-catenin, c-myc, cyclinD1 and DNMT-1 were aberrantly higher in all cell lines we used ( MEC-1,LY8, Jeko-1, Grant519, mino and sp53 Fig.1-A,B), most primary CLL patients (Fig.1-C), and B cell lymphoma tissues (Fig.1-D). The protein expressions of beta-catenin in MEC-1 were higer than primary CLL patients. 0, 0.5, 1.0, 2.0¦ÌM 5-aza-2-deoxycytidine were given to the B cells lines and PBMCs from primary CLL patients for 48h, beta-catenin were found accumulated, but c-myc and cyclinD1 in the downstream were reduced (Fig.2-A,B,C,D). For further understanding of aberrant accumulation ofbeta-catenin, we extracted the nuclear protein of MEC-1, nuclear beta-catenin protein expressions were found decreased and cytoplasmic were increased (Fig.2-E). After 5-aza treatment, the apoptosis rate increased and caspase pathway were activated (Fig.2-A,F). Conclusions: The enhanced expressions of beta-catenin, c-myc, cyclinD1 in the B cell lines and the B cell lymphoma samples indicated the Wnt/beta-catenin was aberrantly activated. After 5-aza treatment with the cell lines (MEC-1, Jeko-1, LY8) and primary CLL cells, the abnormal accumulation of beta-catenin protein was observed which was discrepancy with previous reports, but the decrease of c-myc and cyclinD1 suggested the pathway was inhibited, apoptosis also occurred. The increase of totalbeta-catenin protein was supposed to be an stress reaction of the 5-aza treatment, however, the redundant beta-catenin protein in B cell lymphoma was speculated to be combined with demethylated genes and resulted in dormancy of this pathway. Our results indicated that 5-aza played a demethylation role through Wnt/beta-catenin pathway in B cell lymphoma. The data are of interest in the context of epigenetic-based therapy in B cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

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The Contribution of Proteasome Subunits to Myeloma Cell Viability and Proteasome Inhibitor Sensitivity

Blood ◽

10.1182/blood-2019-127149 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4337-4337

Author(s):

Chang-Xin Shi ◽

Yuan Xiao Zhu ◽

Laura Ann Bruins ◽

Cecilia Bonolo De Campos ◽

William Stewart ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Viability ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Resistance Function ◽

Proteasome Subunits ◽

Underlying Mechanisms

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

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Combination of the IGF-1 Receptor Inhibitor Picropodophylin and the BH3 Mimetic ABT-737 Has Synergistic Anti-Myeloma Activity

Blood ◽

10.1182/blood.v120.21.4010.4010 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4010-4010

Author(s):

Liesbeth Bieghs ◽

Ken Maes ◽

Els Van Valckenborgh ◽

Eline Menu ◽

Hans Erik Johnsen ◽

...

Keyword(s):

Growth Factors ◽

Cell Viability ◽

Cell Lines ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Differential Sensitivity ◽

Expression Ratio ◽

Therapy Strategy ◽

Induced Apoptosis ◽

Bh3 Mimetic

Abstract Abstract 4010 Despite intensive research multiple myeloma (MM) is still an incurable disease. MM cells are strongly dependent on the BM micro-environment where growth factors are secreted. IGF-1 is one of the most important growth factors in MM and thus forms an attractive target for anti-cancer therapy. Previously, we demonstrated that picropodophylin (PPP), an IGF-1R kinase inhibitor, indeed has potent anti-MM effects both on human cells and in the 5T2MM and 5T33MM murine models. However, mice eventually relapsed and showed signs of morbidity. Therefore it would be an attractive approach to combine PPP with other cytotoxic drugs. ABT-737 is a BH3 mimetic that binds with high affinity to Bcl-xL, Bcl-2 and Bcl-w, but not Mcl-1. In MM, elevated expression of the Bcl-2 pro-survival family of proteins, especially Mcl-1 and to a lesser extent Bcl-2, has extensively been shown to cause resistance to drug induced apoptosis in MM cells. Consequently, ABT-737 was shown to have potent anti-MM activity but only on a subset of human cell lines. Only very recently, it was shown that the ABT-737 sensitivity appears to be determined both by the Bcl-2/Mcl-1 expression ratio and the interaction of these anti-apoptotic proteins with Bim. Interestingly, we demonstrated that IGF-1R inhibition reduces the expression of Mcl-1 and Bcl-xL and that IGF-1 down-regulates the expression of Bim. In addition, a protective effect of IL-6 and BMSC against ABT-737 has been reported. Together, all the above suggests that it would be beneficial to combine ABT-737 with agents that target growth factors, like PPP. Here, we investigated the potential synergistic anti-MM effects of PPP and ABT-737 and studied the underlying mechanisms using two human myeloma cell lines (OPM-2 and RPMI-8226) and the murine 5T33MM model. Both PPP and ABT-737 (kindly provided by Abbott Laboratories) alone were found to significantly decrease cell viability and induce apoptosis dose and time dependently as evidenced by a decrease in ATP levels and an increase in the number of AnnexV/7'AAD positive cells. However, in agreement with previous reported data, we observed differential sensitivity to ABT-737 between the cell lines used. Nevertheless, treatment with PPP/ABT-737 synergistically decreased cell viability and induced apoptosis in all cell lines. In addition, by western blot analysis we could observe increased cleavage of caspase- 3,- 9 and PARP. Mechanistically, PPP was found to circumvent the adverse effect of ABT-737 by blocking the ABT-737 induced Mcl-1 expression and increasing the expression of Noxa. Interestingly, while CD138+ 5T33MM cells were more sensitive to PPP and the CD138- cells more sensitive to ABT-737, treatment with PPP/ABT-737 targeted both MM cell subpopulations to an equal extent. Finally, we tested the combination of PPP and ABT-737 in the 5T33MM model in a prophylactic setting. Whereas, vehicle and ABT-737 treated mice exhibited progressive MM growth, PPP, and to a significant greater extent, PPP/ABT-737 reduced the tumor burden and prolonged overall survival (p≤0.001). In conclusion, PPP combined with ABT-737 appears to have synergistic anti-MM activity and might thus be a novel and promising therapy strategy for MM. Disclosures: No relevant conflicts of interest to declare.

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YB-1 Is Activated by IL-7 and Is Overexpressed in Pediatric Pre-B Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1468.1468 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1468-1468

Author(s):

Amina Kariminia ◽

Sabine M Ivison ◽

Vivian Leung ◽

Sandra E. Dunn ◽

Aru Narendran ◽

...

Keyword(s):

Flow Cytometry ◽

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

B Cells ◽

Signaling Pathways ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

B Cell Lymphoma ◽

Fluorescent Intensity

Abstract Abstract 1468 Background: Increased YB-1 expression correlates with poor prognosis, drug resistance and metastasis in several different cancers including B cell lymphoma. Phosphorylation and nuclear localization of YB-1 in response to growth factors leads to increased survival through expression of proteins such as survivin and multidrug resistance protein 1. Until now, its role in leukemia has not been investigated. We hypothesized that YB-1 expression is aberrantly regulated in pediatric pre-B acute lymphoblastic leukemia (pre-B ALL), and that YB-1 may be activated downstream of IL-7. This cytokine facilitates the differentiation and survival of pre-B cells and has been implicated in the drug resistance of pre-B ALL. Methods: YB-1 and IL-7Ra protein expression was investigated by flow cytometry in normal pre-B cells (CD19+CD10+CD20−), and mature B cells (CD19+CD10−CD20+) as well as diagnostic and relapsed pre-B ALL (CD19+CD10+/−). Cell surface and cytoplasmic expression was quantified by mean fluorescent intensity (MFI). Bone marrow from healthy donors was used as a source of normal pre-B cells, while mature B cells were derived from PBMCs; leukemic cells at presentation and relapse were obtained following local IRB approval and informed consent. Activation of YB-1 downstream of IL-7 stimulation (25 ng/ml) was examined in pre-B ALL cell lines or NSG (NOD scid gamma) mice-expanded pre-B ALL by Western blotting using anti-phosphoYB-1(S012). Pre-B ALL cell lines used in these experiments were 697, 380, RCH and RS-4;11. Signaling pathways were investigated by pre-treatment of cells with pharmacological inhibitors followed by Western analyses. For the transient overexpression of YB-1, pEGFP or a pEGFP/YB-1 fusion protein was electroporated into freshly isolated mature B cells (which have a low basal expression of YB-1) and YB-1 and IL-7Ra expression was assessed by flow cytometry after 24 h. Results: While intracellular YB-1 expression was significantly higher in leukemia samples at presentation compared to normal pre-B cells, the highest YB-1 levels were found in relapsed pre-B ALL (see figure, part A). All examined pre-B ALL cell lines had levels comparable to those of the relapse samples. Similarly, surface IL-7Ra (CD127) levels (MFI medians; upper-lower range) were increased in onset (221; 150–286), and relapsed (1840; 651–2030) ALL compared to normal pre-B cells (528; 333–2673). (normal pre-B vs. leukemia at presentation, p<0.001, Mann-Whitney). Overexpression of YB-1-GFP in normal mature B cells resulted in increased expression of IL-7Ra (see figure, part B), suggesting an link between the YB-1 and IL-7 signaling pathways. Activated YB-1 is phosphorylated on S102 and relocated to the nucleus. Addition of IL-7 to pre-B ALL cell lines led to phosphorylation of YB-1 within 30 min. Similar results were shown for patient-derived, NSG mice-expanded pre-B ALL samples. Intracellular immunostaining using Imagestream technology (Amnis) showed that IL-7 treatment of pre-B ALL cell lines increased nuclear YB-1 levels 4-fold. As PI3K and MEK1 are involved in signaling downstream of IL-7, we investigated their role in YB-1 signaling in both pre-B ALL cell lines and NSG-mouse expanded pre-B ALL using pharmacological inhibitors. Western analyses showed that inhibition of PI3K using LY294002 did not prevent IL-7-mediated phosphorylation of YB-1 but the MEK1 inhibitor U0126 did, indicating the involvement of MAPK (see figure, part C). Conclusion: We show that YB-1, which is highly expressed in pediatric pre-B ALL compared to normal pre-B cells, is expressed at even higher levels after relapse. We demonstrate a link between the YB-1 and IL-7 signaling pathways which could offer a novel target for the treatment of refractory leukemia. Disclosures: No relevant conflicts of interest to declare.

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Ibrutinib Sensitizes AML Cells to ROS Inducers Via a BTK-Independent Mechanism

Blood ◽

10.1182/blood.v124.21.2226.2226 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2226-2226 ◽

Cited By ~ 3

Author(s):

Lianne E Rotin ◽

Marcela Gronda ◽

Neil Maclean ◽

Feng-Hsu Lin ◽

Jeff Wrana ◽

...

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Synergistic Effects ◽

Single Agent ◽

B Cell Lymphoma ◽

Fold Increase ◽

Synergistic Cytotoxicity ◽

Btk Inhibitor

Abstract Ibrutinib is a small-molecule Bruton’s tyrosine kinase (BTK) inhibitor yielding impressive clinical responses in B-cell malignancies, where BTK contributes to disease development and progression. We noted that BTK is expressed and constitutively active in acute myeloid leukemia (AML) cell lines and in a subset of AML patients. We therefore sought to determine BTK’s potential role in AML. Using BTK-pY223 expression on immunoblot as a marker of BTK activity, we demonstrated that ibrutinib doses as low as 1μM were sufficient to inhibit BTK activity in the AML cells TEX and OCI-AML2. Yet, these cell lines were insensitive to ibrutinib compared to the B-cell lymphoma Daudi cell line (IC50 10.4μM and 23.7μM versus 1.1μM, respectively). Although inactive as a single agent, we sought to identify drug combinations that sensitized AML cells to pharmacologically relevant concentrations of ibrutinib by conducting a combination chemical screen with our in-house known drug library in TEX and OCI-AML2 cells. According to excess-over-Bliss additivism criteria, we determined that in both cell lines, the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate was the most synergistically cytotoxic, producing an 8-fold reduction in the IC50 of ibrutinib. Synergistic cytotoxicity was also observed in a subset of primary AML cells, but not normal hematopoietic cells. Mechanistically, the combination’s synergistic cytotoxicity resulted from excessive ROS production (>10-fold increase, versus a <2-fold increase with either drug alone in TEX and OCI-AML2), since α-tocopherol addition to combination-treated cells rescued cell viability from 24% to 85% and from 4% to 84% in TEX and OCI-AML2, respectively. Combining ibrutinib with other ROS-inducing agents such as parthenolide and the first-line AML therapy daunorubicin produced similar synergistic effects in AML cells,with α-tocopherol also rescuing cell viability. However, the synergistic effects were likely not related to inhibition of BTK, as knockdown of BTK with shRNA did not sensitize TEX cells to ethacridine or daunorubicin and thus did not recapitulate the effects of ibrutinib. We therefore conclude that the BTK inhibitor ibrutinib lacks single agent anti-AML activity, but synergizes with ROS-inducing agents including daunorubicin by inhibiting targets beyond BTK. Thus, clinical trials of ibrutinib in combination with standard chemotherapy could be warranted in AML. Disclosures No relevant conflicts of interest to declare.

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HIF-1a Regulating the Chemoresistance By Upregulating the Expression of xCT and GCLM in the Diffuse Large B Cell Lymphoma

Blood ◽

10.1182/blood-2019-127612 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5236-5236

Author(s):

Yongqiang Wei ◽

Hong Zeng ◽

Xiaolei Wei ◽

Weimin Huang ◽

Jialin Song ◽

...

Keyword(s):

Drug Resistance ◽

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Response To Treatment ◽

Chop Regimen ◽

Large B Cell Lymphoma ◽

Large B Cell

Background Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-hodgkin lymphoma with great heterogeneity in clinical behavior and response to treatment. Although the addition of rituximab to CHOP regimen has significantly improved the survival of DLBCL, 1/3 will be eventually relapse and progression. HIF1a has been reported to be related with the drug resistance in DLBCL, but the mechanism remains unknown. Methods Two DLBCL cell lines (Riva and SuDHL2) were treated with doxorubicin and HIF-1a inhibitors digoxin, YC-1. The proliferation of these cell lines (Riva and SuDHL2) were measured by MTT and apoptosis were detected by FCM after staining with Annexin V/SYTOX Green. Expression of IF-1α, PHD, xCT, GCLM and apoptosis-related proteins was detected by Western Blot. Results With the increase concentration of doxorubicin treatment, the proliferation of Riva and SuDHL2 cell lines could be gradually inhibited. xCT and GCLM expression were upregulated after treated with doxorubicin and can be reversed by N-acetylcysteine. HIF-1a inhibitors digoxin and YC-1 could inhibited the expression of xCT and GCLM, proliferation and apoptosis induced by doxorubicin. Furthermore, we treated shHIF-1a RNA to significantly reduce the expression of HIF-1a, and the decrease of HIF-1a expression enhanced the proliferation inhibition and apoptosis of lymphoma cells by Dox. Downregulation the expression of HIF-1a by shRNA enhanced the doxorubicin induced apoptosis and inhibited the proliferation in DLBCL cell lines. Conclusions Taken together, our results showed that HIF-1a could regulate the expression of xCT and GCLM and further mediate drug resistance in the diffuse large B cell lymphoma. Disclosures No relevant conflicts of interest to declare.

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Birinapant Enhances Bendamustine-Induced Apoptosis In Activated B Cell-Diffuse Large Cell Lymphoma Cells

Blood ◽

10.1182/blood.v122.21.5150.5150 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5150-5150

Author(s):

Indira D Joshi ◽

Mitchell R Smith

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Single Agent ◽

Annexin V ◽

B Cell Lymphoma ◽

Lymphoma Cell ◽

Low Level ◽

Induced Apoptosis

Abstract Birinapant (TL32711), a Smac mimetic in clinical testing, potently targets Inhibitor of Apoptosis Proteins (IAPs, including cIAPs and XIAP) to unblock intrinsic and extrinsic pathways, enabling caspase-dependent apoptosis via multiple signals. Birinapant also inactivates canonical NF-kB signaling through cIAPs. We investigated the pro-apoptotic effects of birinapant, alone and in combination with bendamustine (BDM), an active lymphoma therapeutic agent, in a panel of B cell lymphoma cell lines representing germinal center/follicular (GC) vs. activated B cell (ABC) subtypes. We hypothesized that the efficacy of this potential combination therapeutic strategy might differ between GC and ABC lymphoma types, as ABC are reported to be NF-kB-dependent. We used the following EBV negative cell lines: WSU-FSCCL t(14:18)+ follicular lymphoma (FL), FC-TxFL2 t(14:18)+ transformed FL, and SU-DHL4 GC-type diffuse large B cell lymphoma (DLBCL) as examples of GC origin lymphomas. U2932 and TMD8 cell lines represent ABC-type DLBCL. Apoptosis was determined by annexin V staining and confirmed by caspase-3 activation, each assessed by flow cytometric methods following 48 h incubation. Birinapant had little effect (<5% annexin V+ cells) as a single agent on any of these B cell lymphoma cell lines at ≤ 100 nM, though a low level of apoptosis (7-12% annexin V+ cells) was detectable at 10-20 µM in GC types. Addition of birinapant 30-60 minutes prior to BDM did not further enhance the already high level (>50% annexin V+) of apoptosis induced by 10 uM BDM in WSU-FSCCL and FC-TxFL2, and only slightly enhanced the low level of BDM-induced apoptosis in the GC DLBCL cell line DHL-4 (to 10-15%). In the ABC DLBCL cell lines, however, whereas 10uM BDM induced <5% annexin V+ cells for U2932 and 10-15% for TMD8, addition of 100 nM birinapant 30-60 minutes prior to 10 uM BDM induced 35-40% annexin V+ cells in each of these ABC-DLBCL cell lines. This enhancement was schedule-dependent, not observed when birinapant was added after BDM. Thus, the cell lines representing FL and transformed FL are sensitive to BDM at clinically-achievable concentrations, without further enhancement by birinapant. The 3 DLBCL lines were relatively insensitive to BDM compared with FL cells, but BDM-induced apoptosis was markedly enhanced when birinapant was added before (but not after) BDM in the 2 ABC type DLBCL lines. Further explorations into the mechanism of birinapant sensitization of ABC-DLBCL to BDM, issues of dose and schedule, and role of NF-kB-dependency are ongoing. These data suggest that therapeutic trials of BDM plus birinapant would be of interest in ABC type DLBCL. Disclosures: No relevant conflicts of interest to declare.

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Using the Iron Chelator Deferasirox to Overcome Drug Resistance in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2021-150443 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4364-4364

Author(s):

Aladin Samara ◽

Saar Shapira ◽

Ido Lubin ◽

Pia Raanani ◽

Galit Granot

Keyword(s):

Drug Resistance ◽

B Cell ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cell Lymphoma ◽

Synergistic Effects ◽

Iron Chelator ◽

Mantle Cell ◽

Resistant Cells

Abstract Mantle cell lymphoma (MCL) is a difficult-to-treat B-cell malignancy characterized by the t(11,14) translocation, resulting in cyclin D1 (CD1) overexpression. In addition to CD1 overexpression, pathways such as the B-cell receptor, PI3K/AKT/ GSK3β, NFκB and Wnt have been reported to be deregulated in MCL. Despite advancements in MCL treatment, most patients still relapse. Although the introduction of ibrutinib to relapsed/refractory MCL significantly improved the outcome of MCL patients, ibrutinib resistance has become a clinical obstacle. MCL treatments are thus pursued by studying novel agents with a broad spectrum of targets or by rationally combining existing therapies aiming for synergistic antitumor activities. Deferasirox (DFX) is a clinically approved iron chelator with only few side effects. DFX has been reported to exert anti-tumoral and synergistic effects in several types of cancers by affecting a multitude of targets. We have previously shown that DFX exerts a vigorous anti-tumoral effect via growth inhibition and induction of apoptosis in MCL cells through ROS elevation, triggering of oxidative stress, induction of DNA damage, modulation of PI3K/AKT/GSK3β signaling and most importantly by elimination of CD1 expression. The capacity of DFX to affect a multitude of targets establishes a solid basis for a possible synergistic interaction with other drugs, such that may overcome drug resistance in MCL. In this study we focused on assessing the efficiency of DFX combined with the established therapeutic-agents etoposide, cytarabine and ibrutinib in MCL cell-lines. We found that DFX synergizes with etoposide, cytarabine and ibrutinib, prompting remarkable anti-tumoral effects in MCL cells with combination index (CI) values < 1. Interestingly, the DFX-drug combinations achieved synergism regardless of the innate sensitivity of the cell-lines to the treatment: ibrutinib-resistant cells restored their sensitivity to the drug when it was combined with DFX. In addition, we found that the sensitivity of MCL cells towards the drugs correlated with the drugs ability to induce CD1 degradation. In agreement, DFX co-treatment enhanced CD1 degradation, especially in resistant cells. We show here that DFX is a putative promising drug-sensitizing agent for the treatment of MCL. DFX-co-treatment not only enhances the efficacy of the tested drugs, but also restores the anti-proliferative activity of the drugs in resistant MCL cells. To the best of our knowledge, this study is the first to provide evidence on the potential of DFX to overcome drug resistance in MCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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