Stromal Cell-mediated Drug Resistance Research Articles

Clarithromycin overcomes stromal cell-mediated drug resistance against proteasome inhibitors in myeloma cells via autophagy flux blockage leading to high NOXA expression.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

Bone Marrow Stromal Cell Mediated Drug Resistance in AML Cells: Role of Extracellular Vesicles and AKT Signaling

Accumulating evidence suggests that bone marrow stromal micro-environment act as sanctuary site for acute myeloid leukemia (AML) cells and affords protection from conventional chemotherapy agents. Recently, there has been considerable interest in extracellular vesicles (EVs) and their role in intercellular communication. We had previously reported that there is significant stromal cell mediated drug resistance in acute promyelocytic leukemia (APL) which was predominantly driven by up-regulation of the NF-kB pathway in the malignant cells (Ganesan S et al, Leukemia 2016). Though there are reports of similar stromal cell mediated protection against chemotherapy in non-M3 AML cells, there is limited data on the role EVs play, if any, in inducing such chemotherapy resistance. We undertook a preliminary study to evaluate the role of EVs in inducing resistance to chemotherapeutic agents in AML.We demonstrated that direct stromal co-culture experiments with AML cell lines as well as primary AML cells showed a significant stromal mediated protective effect against both cytosine arabinoside (AraC) and daunorubicin (DNR) [U937: AraC p < 0.01, DNR p < 0.001 (n=5); primary AML cells: AraC p < 0.01, DNR p < 0.001 (n=26)]. Gene expression profiling (GEP) analysis of AML cell line U937 co-cultured with HS-5 revealed a significant enrichment of genes involved in PI3K-AKT signaling pathway (p < 0.0001) followed by NF-kB pathway genes. It has been previously reported that on direct co-culture with stromal cells there is activation of the PI3K-AKT pathway in AML cells leading to drug resistance (Tabe Y et al. Cancer Res 2007). It is also well recognized that PI3K-AKT pathway can drive NF-kB signaling, which is consistent with our earlier reported observation in APL. We hypothesize that this effect of PI3K-AKT activation could be mediated by EVs without direct malignant and stromal cell interaction as well. Towards this, we isolated stromal EVs as previously described (Suzanne M et al, Blood 2016). The size and concentration of EVs was characterized by NanoSight and flow cytometry (data not shown).We demonstrated an activation of PI3K-AKT signaling by showing phosphorylation of AKT (P-AKTser473) in U937 cells when treated with stromal EVs (figure 1A). We further demonstrated that stromal EVs were able to mediate a protective effect against AraC and DNR similar to that seen on direct co-culture with HS-5 cells [AraC p < 0.01, DNR p < 0.01 (n=3)]. This was further confirmed by looking at the pathway genes using Q-PCR, where an increased expression of PI3K-AKT signaling was observed in AML cells upon treatment with stromal EVs. The activation PI3K signaling was also confirmed by showing down regulation of PTEN [p < 0.01 (n=5)] which is a negative regulator of PI3K-AKT signaling. Further, to demonstrate the role of stromal EVs induced PI3K-AKT signaling in drug resistance, we treated the leukemic cells with a small molecule inhibitor of PI3 kinase (LY294002-10uM) along with AraC and DNR in the presence of stromal EVs. We noted that inhibiting PI3K-AKT signaling along with AraC or DNR was able to overcome the resistance mediated by stromal EVs (figure 1B). The mechanism of inducing apoptosis by LY294002 along with DNR or AraC (in the presence of stroma or stromal EVs) probably involves a TP53 dependent apoptosis pathway as evidenced through upregulation of TP53 and its target apoptotic genes such as BAD, CASP9 and PUMA (AraC- figure 1C, DNR - data not shown).Our study thus illustrates the complexity of cellular crosstalk between leukemic cells and its microenvironment in inducing drug resistance. This resistance can be mediated by non-contact setting via stromal EVs through up regulation of PI3K-AKT signaling in myeloid leukemic cells. Our data highlights the need for additional studies that are required to explore and characterize in detail the cargo of these EVs and the mechanisms by which they mediate drug resistance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Microrna-494 Activation Suppresses Bone Marrow Stromal Cell-Mediated Drug Resistance in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is not sensitive to chemotherapy partially because of the protection of AML cells by mesenchymal stromal cells (MSCs). Our previous studies found that MSCs protected AML cells from apoptosis through the c-Myc-dependent pathway. However, the mechanism by which MSCs regulate c-Myc in AML cells is still unknown. To elucidate the mechanism, we performed microRNA array analysis of AML cell lines and validated by TaqMan realtime PCR. The results showed that the expression of microRNA-494 (miR-494) in AML cells after coculture with MSCs was down-regulated. Reporter gene analysis confirmed miR-494 as one of the regulators of c-Myc. In the coculture system, activation of miR-494 in AML cells suppressed proliferation and induced apoptosis of AML cells in vitro. After addition of mitoxantrone to the coculture system, the proliferation of AML cells with miR-494 activation was suppressed more than that of control cells. After subcutaneous injection of AML cell lines in combination with MSC, tumor growth was suppressed in mice injected with miR-494-overexpressing AML cells. The rate of tumor formation was even lower after mitoxantrone treatment in the miR-494 overexpressing group. Moreover, miR-494 activation resulted in a decrease of leukemic cell counts in peripheral blood and bone marrow and prolonged survival in mice injected with miR-494-overexpressing AML cellls and MSCs compared to the control mice. Our results indicate that miR-494 suppresses drug resistance in AML cells by down-regulating c-Myc through interaction with MSCs and that miR-494 therefore is a potential therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Granulocyte colony-stimulating factor inhibits CXCR4/SDF-1α signaling and overcomes stromal-mediated drug resistance in the HL-60 cell line.

Combining cytarabine, aclarubicin and granulocyte colony-stimulating factor (G-CSF) has demonstrated marked efficacy in the treatment of elderly and relapsed/refractory patients with acute myeloid leukemia (AML); however, the role of G-CSF remains poorly understood. The present study aimed to investigate the ability of G-CSF to overcome stromal-mediated drug resistance and the underlying molecular mechanism. Two types of co-culture models were established in the HS-5 human bone marrow/stromal and HL-60 human promyelocytic leukemia cell lines, in order to imitate the interactions between stromal and leukemia cells in vitro, which is mediated by the stromal cell-derived factor (SDF)-1α signaling axis. In the present study, HL-60 cells were attracted and adhered to HS-5 cells using migration assay and flow cytometry, respectively; however, these interactions were inhibited by treatment with G-CSF and/or the C-X-C chemokine receptor type 4 (CXCR4) antagonist, AMD3100. Co-culture with HS-5 cells, including direct and indirect contact, protected HL-60 cells against spontaneous apoptosis or drug-induced apoptosis; however, these protective effects were disrupted by treatment with G-CSF and/or AMD3100. Notably, G-CSF and/or AMD3100 did not alter cell viability or apoptosis when HL-60 cells were cultured with medium alone. In addition, G-CSF significantly reduced the expression levels of surface CXCR4 protein, total CXCR4 protein and CXCR4 mRNA, and significantly upregulated the expression of microRNA (miR)-146a. Conversely, AMD3100 significantly reduced surface CXCR4 expression levels, but not the total CXCR4, CXCR4 mRNA or miR-146a expression levels. The results of the present study suggested that interfering with the CXCR4/SDF-1α signaling axis via G-CSF inhibited the migration and adhesion of HL-60 cells to HS-5 cells and eliminated HS5 cell-mediated protective effects. Furthermore, G-CSF administration reduced CXCR4 expression levels by upregulating the expression of miR-146a, whereas AMD3100 appeared to be predominantly dependent on receptor internalization. Therefore, a G-CSF/miR-146a/CXCR4 pathway may explain how G-CSF inhibits CXCR4/SDF-1α signaling and overcomes stromal cell-mediated drug resistance in acute myeloid leukemia.

Role of CXCR4/STAT3 in mesenchymal stromal cell-mediated drug resistance of acute leukemia cells

目的探讨CXCR4/STAT3在骨髓基质细胞介导的急性髓系白血病(AML)细胞耐药中的作用。方法将AML细胞系U937、KG1a和原代AML细胞与健康供者来源的骨髓基质细胞共培养，单独培养的细胞作为对照组。通过膜联蛋白Ⅴ (Annexin Ⅴ)／碘化丙锭(PI)双染色法比较共培养前后AML细胞对米托蒽醌诱导的凋亡的差异，并通过流式细胞术、实时定量PCR及Western blot法检测共培养前后AML细胞CXCR4、磷酸化STAT3(p-STAT3)蛋白的表达；在培养体系中加入STAT3的特异性抑制剂Cucurbitacin Ⅰ或CXCR4拮抗剂AMD3100，检测AML细胞对米托蒽醌诱导的凋亡的变化，并观察AMD3100对p-STAT3表达的影响。结果U937、KG1a细胞与骨髓基质细胞共培养后，对米托蒽醌诱导的凋亡率均低于对照组[U937：(20.08±1.53)%对(45.33±1.03)%，P=0.004; KG1a：(25.60 ± 1.82)%对(40.33±3.29)%，P=0.003]。共培养后，Western blot方法检测发现AML细胞p-STAT3的蛋白表达明显上调，流式细胞术和Western blot检测结果显示CXCR4蛋白表达显著上调，实时定量PCR检测显示CXCR4 mRNA水平明显增高；STAT3的特异性抑制剂Cucurbitacin Ⅰ或CXCR4拮抗剂AMD3100加入共培养体系后，原代AML细胞对米托蒽醌诱导的凋亡率较对照组显著升高，差异均有统计学意义(P值均<0.05)，且加入AMD3100后AML细胞p-STAT3蛋白的表达显著下降。结论AML细胞与骨髓基质细胞共培养可导致p-STAT3、CXCR4蛋白表达的上调，从而导致AML细胞对化疗药物的耐药；针对STAT3、CXCR4的联合靶向治疗可能为AML的治疗提供新思路。

Abstract 4881: Targeting the CXCR4 chemokine receptor thwarts stromal cell mediated drug resistance in B-cell acute lymphoblastic leukemia (B-ALL)

Abstract CXCR4 inhibitors Plerixafor (small molecule) and BKT140 (peptide) interfere with CXCR4 activation, signaling and bone marrow homing of leukemia cells. Adhesion of B-ALL cells to bone marrow stromal cells (BMSC) through CXCR4/CXCL12 protects leukemia cells from cytotoxic drugs, and causes cell adhesion-mediated drug resistance (CAM-DR). Due to the critical role of the CXCR4/CXCL12 axis in B-ALL cell adhesion to bone marrow stromal cells (BMSC), we hypothesize that targeting CXCR4 will attenuate CAM-DR in B-ALL. All tested B-ALL cell lines and xenografts were found to be positive for surface expression of CXCR4, using 12G5 mAbs. Pre-treatment with Plerixafor (10μg/ml) or BKT140 (10μg/ml) significantly inhibited the spontaneous migration of B-ALL cells beneath BMSCs in vitro (Pseudoemperipolesis/PEP) to levels that were 61.4 ± 3.3% or 53.2 ± 5.6%, of respective controls (mean ± SEM, p&amp;lt;0.05). B-ALL cell line and xenograft B-ALL cell chemotaxis towards CXCL12 (100ng/ml) was also abrogated by pre-incubation with 10μg/ml Plerixafor (mean ± SEM, 26.7 ± 4.9% of control, p&amp;lt;0.05) and 10μg/ml BKT140 (mean ± SEM, 18.3 ± 4.7% of control, p&amp;lt;0.05). In order to better dissect CXCR4 function in B-ALL, we established two CXCR4 knock out cell lines, TANOUE-CXCR4-KO and NALM6-CXCR4-KO, using Zinc Finger Nuclease and CRISPR Cas9 technologies respectively. Chemotaxis to 100ng/ml CXCL12 and PEP beneath BMSCs for TANOUE-CXCR4-KO was 10.3 ± 0.48%, n=3 (p&amp;lt;0.05) and 50.06 ± 2.5%, n=4 (p&amp;lt;0.05) respectively, in comparison to TANOUE wild type control without any drug treatment. For NALM6-CXCR4-KO chemotaxis and PEP was 43.1%, n=1 and 41.8 ± 1.9%, n=4 (p&amp;lt;0.05) respectively in comparison to NALM6 wild type control without any drug treatment. CAM-DR experiments with ICN12 xenograft-BMSC co-culture showed significantly enhanced cytotoxicity when cells were treated with a combination of 100nM Dexamethasone (DEX) and 10μg/ml Plerixafor (66.1 ± 5.6% viability, n=3, p&amp;lt;0.05) or 10μg/ml BKT140 (59.7 ± 6.07% viability, n=3, p&amp;lt;0.05) as compared to DEX alone (87.3 ± 2.6% viability) at 48 hours. Similarly, pre-incubation of NALM6 cells with 10μg/ml Plerixafor/BKT140 showed significantly enhanced cytotoxicity (57.93±3.26%, n=3, p&amp;lt;0.05 and 54.2±3.26%, n=3, p&amp;lt;0.05 respectively) in comparison to DEX alone (72.38±0.25%, n=3). BMSCs rescued NALM6 cells from DEX induced cytotoxicity but failed to rescue NALM6-CXCR4-KO cells indicating that CXCR4 is the central mechanism used by stromal cells to provide drug resistance to ALL cells. Collectively, these findings provide a rationale for clinical targeting of CXCR4 in patients with B-ALL. Citation Format: Shubhchintan Randhawa, Dipanjan Ghosh, Amnon Peled, Richard E. Davis, Jan A. Burger. Targeting the CXCR4 chemokine receptor thwarts stromal cell mediated drug resistance in B-cell acute lymphoblastic leukemia (B-ALL). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2014-4881

Role of c-Myc in mesenchymal stromal cell-mediated drug resistance in acute leukemia cells

To explore the role of c-Myc in mesenchymal stromal cell-mediated drug resistance and elucidate the molecular mechanism of acute myeloid leukemia (AML) from the version of tumor microenvironment. AML cell lines U937 and KG1a were co-cultured with mesenchymal stromal cells (MSC) from bone marrow of healthy donors between January to March 2012. The AML cell lines plated alone was cultured as controls. Apoptosis induced by mitoxantrone was measured by flow cytometry and Annexin V/PI double and 4'-6-diamidino-2-phenylindole (DAPI) staining. And c-Myc protein was detected by Western blot under both culturing conditions. After a pre-treatment of c-Myc inhibitor 10058-F4, the apoptosis of AML cell was also evaluated. Apoptosis of AML cells (U937 and KG1a) significantly decreased during co-culturing with MSC (9.88% ± 1.53% vs 42.83% ± 2.03%, P = 0.004;20.60% ± 2.87% vs 42.53% ± 5.29%, P = 0.030). Drug resistance was implicated. The co-culturing of AML cells with MSC significantly induced an up-regulation of c-Myc. The inhibition of c-Myc with 10058-F4 could induce apoptosis of AML cells. After an addition of 10058-F4 into the co-culture system, the apoptotic rate of KG1a cells significantly increased from 23.87% ± 1.55% to 57.23% ± 3.88% (P = 0.009). Similarly the apoptotic rates spiked from 16.07% ± 2.11% to 53.47% ± 4.08% in U937 cells (P = 0.004) to overcome the stromal cell-mediated drug resistance. The co-culturing of AML cells and MSC induces an up-regulation of c-Myc protein so as to cause the emergence of chemoresistance. Therefore targeting c-Myc protein may provide a novel therapeutic strategy of AML.

Abstract 3378: The rGel/BLyS fusion toxin can overcome chemoresistance in malignant B cells..

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive sub-type accounting for 30-40% of non-Hodgkin's lymphoma. Approximately 95% of B cell lymphoma expresses CD20 and 26% of relapsed B-cell lymphoma patients demonstrate reduced CD20 expression on tumor cells after treatment with the anti-CD-20 antibody rituxumab. This result suggests that novel agents that can overcome rituximab resistance may be an excellent candidate for the treatment of DLBCLs which are resistant to rituximab. MCL and ABC-DLBCL cell lines (SP53, Mino, OCI-Ly3, OCI-Ly10 and U2932) were resistant to rituximab whereas GC-DLBCL cell lines (SUDHL-4 and SUDHL-6) were very sensitive to rituximab. To understand the mechanism of rituximab resistance, we developed rituximab-resistant SUDHL-6 cell line. Compared with parental rituximab-sensitive cells (SUDHL-6-S, IC50=0.2 μg/ml), rituximab-resistant cells (SUDHL-6-R, IC50 &amp;gt; 1,000 μg/ml) demonstrated ∼5,000 fold resistance. The rituximab-sensitive cells formed single-cell suspensions whereas resistant cells demonstrated cell aggregates. We previously generated a highly cytotoxic fusion toxin designated rGel/BLyS for receptor-mediated delivery of the rGel toxin to malignant B cells. The rituximab-sensitive (SUDHL-6-S) and resistant (SUDHL-6-R) both cells showed similar IC50 values (150 nM) for rGel/BLyS. In contrast, intrinsically rituximab-resistant cell lines (SP53, Mino, OCI-Ly3, OCI-Ly10 and U2932) were also sensitive to rGel/BLyS. Treatment with rituximab specifically induced down-regulation of p-Akt (Ser473), Akt, and Syk and activation of caspase-9, caspase-3 and PARP in a dose-dependent manner in rituximab-sensitive cells (SUDHL-6-S) but not rituximab-resistant cell lines (SUDHL-6-R, OCI-Ly3, or OCI-Ly10). The interactions of marrow stromal cells (MSCs) with tumor cells appear to be important in the development of tumor drug resistance through complex cytokine and adhesion-mediated mechanisms. Treatment with rGel/BLyS induced down-regulation of p-Akt, Akt, Syk, interleukin-6 receptor, Mcl-1, and c-Myc in rituximab-resistant DLBCL cells in both the absence and presence of MSCs. In addition, treatment of cells with rGel/BLyS induced apoptosis through caspase-9 activation, caspase-3 activation, and PARP cleavage. These results suggest that the Akt pathway is involved in rituximab resistance and rGel/BLyS can overcome stromal cell-mediated drug resistance which is a factor reducing the effectiveness of other conventionally-used agents for the treatment of DLBCLs which are resistant to rituximab. This research was conducted, in part, by the Clayton Foundation for Research. Citation Format: Mi-Ae Lyu, Lawrence H. Cheung, John W. Marks, Michael G. Rosenblum. The rGel/BLyS fusion toxin can overcome chemoresistance in malignant B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3378. doi:10.1158/1538-7445.AM2013-3378

The JAK1/2 Inhibitor Ruxolitinib Kills Malignant Plasma Cells and Is Synergistic with PI3K and mToR Inhibitors

Abstract 1844Malignant plasma cell growth and survival in multiple myeloma (MM) is regulated by cytokines produced in the tumor environment. Specifically, IL-6 plays a key role by activating important signaling pathways through its gp130 receptor associated Janus kinases (JAK). Ruxolitinib (INC424/INCB018424; Novartis/Incyte) is the first small molecule JAK inhibitor approved for the treatment of patients with myelofibrosis. It is a potent inhibitor of both JAK1 and JAK2 and has an approx. 6-fold selectivity against Tyk2 and marked selectivity against JAK3 (more than 130-fold) and additional kinases. The aim of our study was to evaluate the effects of ruxolitinib on malignant plasma cells as well as its activity in combination with other pathway inhibitors.Ruxolitinib activity was evaluated in MTS-based colorimetric cell growth assays or by [3]H-thymidine uptake. IC50 concentrations and combination index (CI) were calculated with CalcuSyn (Biosoft). Evaluating seven human plasma cell lines, ruxolitinib showed a strong cytotoxic activity on the only IL-6 dependent line INA-6 (IC50 0.23 μM). Complete growth inhibition was achieved at 1 μM, even in the presence of bone marrow stromal cells, whereas stromal cell viability and IL-6 production, as measured by specific ELISA, were maintained. Consistent with the dose-dependent inhibition of IL-6 induced STAT3 phosphorylation, apoptosis was induced, resulting in 39% and 63% annexin V-positive cells in the presence of 1 μM ruxolitinib after 48 or 72 hours, respectively. Likewise, significant growth inhibition was seen in purified tumor cells from a patient with plasma cell leukemia that were stimulated with IL-6 (IC50 0.16 μM), and in a LIF-responsive tumor subline of INA-6 (IC50 0.12 μM). In contrast, autonomously growing MM cell lines were not directly inhibited by ruxolitinib, pointing to the kinase specificity of the drug. However, IL-6 mediated drug resistance can be reversed as shown in dexamethasone-sensitive MM1.S cells.Simultaneous inhibition of JAKs with additional signaling pathways that may be activated in myeloma cells by mutations and/or cytokines such as insulin-like growth factor-1, is hypothesized to result in increased cytotoxicity. In INA-6 cells, p44/p42 MAPK activation due to mutated N-Ras and phosphorylation of S6 protein, a downstream target of the PI3K/AKT/mToR pathway, were not abrogated by ruxolitinib. Using combinations of ruxolitinib with inhibitors of PI3K (Ly294002, NVP-BKM120) and mToR (rapamycin), synergistic effects were achieved with a combination index (CI) <1 at the effective dose levels ED50, ED75 and ED90. Other combinations are currently under evaluation.In conclusion, ruxolitinib has strong direct cytotoxic activity against malignant plasma cells that are dependent on JAK/STAT pathway activation. The rationale exists to combine it with inhibitors of complementary pathways and other drugs to potentiate its activity and overcome cytokine or stromal cell mediated drug resistance. Thus, ruxolitinib, as a generally well tolerated drug, may offer therapeutic options for patients with MM. Clearly, the identification of molecular markers may be helpful to assess its precise use alone or in combination, and to select for patients who will benefit from such a treatment. Disclosures:No relevant conflicts of interest to declare.

Chemokine and Chemokine Receptor Expression in Kidney Tumors: Molecular Profiling of Histological Subtypes and Association With Metastasis

Molecular characterization of renal cell carcinoma may help differentiate benign oncocytoma from malignant renal cell carcinoma subtypes and predict metastasis. Chemokines, eg IL-8 and chemokine receptors such as CXCR4 and 7, promote inflammation and metastasis. SDF-1 is a CXCR4 and 7 ligand with 6 known isoforms. We evaluated the expression of these chemokines and chemokine receptors in kidney specimens. Using quantitative polymerase chain reaction we measured mRNA levels of IL-8, CXCR4 and 7, and SDF1 isoforms α, β and γ in a total of 166 specimens from 86 patients, including 86 tumor samples and 80 matched normal kidney samples. Mean ± SD followup was 18.9 ± 12 months (median 19.5). Renal cell carcinoma specimens included the clear cell, papillary and chromophobe subtype in 65, 10 and 5 cases, respectively, and oncocytoma in 6. A total of 17 cases were positive for metastasis. Median CXCR4 and 7, and SFD1-γ levels were increased twofold to tenfold. SDF1-α and β were unchanged or lower in clear cell renal cell carcinoma and papillary tumors than in normal tissue. Median SDF1-γ, IL-8, and CXCR4 and 7 were increased threefold to fortyfold in chromophobe tumors compared to oncocytoma. CXCR4 and 7 were increased in tumors less than 4 cm (mean 3,057 ± 2,230 and 806 ± 691) compared to oncocytoma (336 ± 325 and 201 ± 281, respectively, p ≤0.016). On multivariate analysis CXCR4 (p = 0.01), CXCR7 (p = 0.02) and SDF1-β (p = 0.005) were independently associated with metastasis. Combined CXCR7 plus SDF1-α and CXCR7 plus IL-8 markers showed the highest sensitivity (71% to 81%) and specificity (75% to 80%) of all individual or combined markers. Chemokines and chemokine receptors differentiate renal cell carcinoma and oncocytoma. Combined SDF1-α plus CXCR7 and IL-8 plus CXCR7 markers have about 80% accuracy for predicting renal cell carcinoma metastasis.

Abstract 1758: Influence of stromal microenvironment on rGel/BLyS-induced cytotoxicity in diffuse large B-cell lymphoma cells

Abstract Diffuse large B cell lymphoma (DLBCL) is an aggressive sub-type accounting for 30-40% of non-Hodgkin's lymphoma. Despite recent advances in the understanding of the molecular and cellular basis for their pathogenesis, these tumors are still associated with poor response to treatment and a fatal outcome. The interactions of marrow stromal cells (MSCs) with tumor cells appear to be important in the development of tumor drug resistance through complex cytokine and adhesion-mediated mechanisms. We previously generated a highly cytotoxic growth factor fusion toxin designated rGel/BLyS for receptor-mediated delivery of the rGel toxin to malignant B cells. This fusion toxin specifically targets malignant B cells expressing the BLyS receptors BAFF-R, TACI, and BCMA. In this study, we examined the impact of marrow stromal cells (MSCs) on rGel/BLyS-induced cytotoxicity in DLBCL cells. Treatment of tumor cell lines with dexamethasone and doxorubicine in the presence of MSCs was found to suppress the cytotoxic effect. In contrast, in vitro treatment with rGel/BLyS showed specific cytotoxic effects to DLBCL cell lines (IC50 ∼ 1-10 nM) in absence of MSCs or in presence of MSCs. At these doses, we found no cytotoxicity to MSCs. Treatment with rGel/BLyS induced down-regulation of Mcl-1, c-Myc, Bcl-xL, X-IAP, and Bcl-2 in DLBCL cells in absence of MSCs or in presence of MSCs. We also found that rGel/BLyS induced down-regulation of interleukin-6 receptor in DLBCL cells in absence of MSCs or in presence of MSCs. In addition, treatment of cells with rGel/BLyS induced apoptosis through caspase-9 activation, caspase-3 activation, and PARP cleavage. Our results demonstrate that rGel/BLyS can overcome stromal cell-mediated drug resistance which is a factor reducing the effectiveness of other conventionally-used agents for the treatment of this disease. This suggests that rGel/BLyS may be an excellent candidate for the treatment of DLBCLs which are resistant to conventional chemotherapeutic regimens. This research was conducted, in part, by the Clayton Foundation for Research and by Translational Research Award (LLS 6234-07) from the Leukemia and Lymphoma Society. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1758. doi:10.1158/1538-7445.AM2011-1758

Bryostatin Enhances the Cytotoxic Effects of Anti-CD22 Immunotoxins in CLL by Two Distinct Mechanisms: Implications for a Sequential Therapy.

Abstract 3429Poster Board III-317Recently, treatment of CLL has been improved by the addition of Rituximab, a monoclonal antibody directed against CD20. Although Rituximab displays modest activity when used as monotherapy, the combination of Rituximab and chemotherapy has demonstrated impressive efficacy in CLL. However, even though remissions can be achieved in the majority of patients, CLL still remains an incurable disease for most patients. Therefore, alternative treatment options are needed. BL22 is a recombinant protein composed of the variable region of a monoclonal antibody, which binds to CD22 on the surface of normal and malignant B-cells and of PE38, a truncated Pseudomonas exotoxin. Previously, we demonstrated that BL22 induced cell death in CLL, involving the intrinsic apoptotic pathway. However, apoptosis induction correlates with expression of CD22 on the surface of CLL cells and was only moderate in CD22low expressing cells. The aim of this study was to increase BL22 cytotoxicity by modulating the surface expression of CD22 on CLL cells. Bryostatin is a macrocyclic lactone which structurally mimics the PKC-activating second messenger diacylglycerol. This PKC modulator has demonstrated anti-leukemic effects in CLL by itself in vivo. Here we show that Bryostatin up-regulates the expression of CD22 on CLL cells in a PKC dependent pathway, thereby increasing the cytotoxic effects of BL22. However, PKC-mediated up-regulation of Mcl-1 attenuated the anti-leukemic effects of BL22. We unravel that CD22 and Mcl-1 up-regulation by Bryostatin occurs in a dose dependent manner. Importantly, higher doses of Bryostatin prevented the undesirable up-regulation of Mcl-1 by inhibition of PKC in spite of CD22 up-regulation. Therefore, combined treatment of BL22 and Bryostatin seems to be a promising approach to enhance the cytotoxic effects of the immunotoxin by modulating PKC activity and CD22 up-regulation. In addition, our data provide evidence for a sequential therapy with both drugs, since CLL cells can be primed with Bryostatin. After a single dose of Bryostatin, PKC-modulation lasts for several days, allowing to administer BL22 at later time points, thereby decreasing the risk of potential drug interactions. Finally we demonstrate that stromal cell mediated drug resistance in CLL can be overcome by Bryostatin/ BL22 therapies. Conclusively, Bryostatin enhances the anti-leukemic effects of BL22 in CLL. Clinical trails are needed to prove if such an approach has clinical impact on the treatment and prognosis of B-CLL. DisclosuresNo relevant conflicts of interest to declare.

Isoform-selective phosphoinositide 3′-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell–mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach

Phosphoinositide 3-kinases (PI3Ks) are among the most frequently activated signaling pathways in cancer. In chronic lymphocytic leukemia (CLL), signals from the microenvironment are critical for expansion of the malignant B cells, and cause constitutive activation of PI3Ks. CXCR4 is a key receptor for CLL cell migration and adhesion to marrow stromal cells (MSCs). Because of the importance of CXCR4 and PI3Ks for CLL-microenvironment cross-talk, we investigated the activity of novel, isoform-selective PI3K inhibitors that target different isoforms of the p110-kDa subunit. Inhibition with p110alpha inhibitors (PIK-90 and PI-103) resulted in a significant reduction of chemotaxis and actin polymerization to CXCL12 and reduced migration beneath MSC (pseudoemperipolesis). Western blot and reverse phase protein array analyses consistently demonstrated that PIK-90 and PI-103 inhibited phosphorylation of Akt and S6, whereas p110delta or p110beta/p110delta inhibitors were less effective. In suspension and MSC cocultures, PI-103 and PIK-90 were potent inducers of CLL cell apoptosis. Moreover, these p110alpha inhibitors enhanced the cytotoxicity of fludarabine and reversed the protective effect of MSC on fludarabine-induced apoptosis. Collectively, our data demonstrate that p110alpha inhibitors antagonize stromal cell-derived migration, survival, and drug-resistance signals and therefore provide a rational to explore the therapeutic activity of these promising agents in CLL.

The IL-6 receptor antagonist SANT-7 overcomes bone marrow stromal cell-mediated drug resistance of multiple myeloma cells.

The bone marrow micro-environment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is IL-6. Therefore, the development and testing of substances that interfere with IL-6 or IL-6 receptor (IL-6R) function might have therapeutic value for the treatment of MM. We analyzed the effect of the IL-6R antagonist SANT-7 on growth and survival of the IL-6--dependent MM cell lines INA-6 and XG-1 as well as primary MM cells from 7 patients co-cultured with bone marrow stromal cells (BMSCs). In particular, we were interested in whether SANT-7 enhances the growth-inhibitory effects of dexamethasone (Dex) and all-trans-retinoic acid (ATRA). None of the drugs when tested as a single substance, including SANT-7, induced major growth inhibition if MM cells were co-cultured with primary human BMSCs. However, when Dex and ATRA were given in combination with SANT-7, strong growth inhibition was achieved in cell lines and primary MM cells. This effect was due to cell-cycle arrest and induction of apoptosis.