Stroma-induced Resistance Research Articles

Bone marrow stroma‐mediated resistance to FLT3 inhibitors in FLT3‐ITD AML is mediated by persistent activation of extracellular regulated kinase

A consistent pattern of response has been observed when FMS-like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3- internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3-ITD AML blasts co-cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti-apoptotic effect is mediated through a combination of direct cell-cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine-activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal-mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3-ITD AML.

Abstract 4962: Stroma-induced molecules and chemotherapy resistance in acute myeloid leukemia cells.

Abstract Overall objective: Acute myeloid leukemia (AML) is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Once a child relapses, the survival rate is very low. Therefore new, rational therapies for AML are desperately needed. Accumulating evidence shows that the bone marrow stroma protects a subset of leukemia cells from chemotherapy, eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in AML. Methods, Results & Conclusions: We used a human bone marrow stromal cell line, HS-27A and two human AML cell lines, THP-1 and NB-4. HS-27A cells provide important contact-dependent interactions with AML cells and secrete only a few soluble factors. Our HS-27A cells constitutively express mOrange, to allow discrimination from AML cells after co-culture. We performed co-culture experiments to determine the ability of stromal cells to confer resistance to chemotherapy. THP-1 and NB-4 cells were cultured alone or co-cultured with HS-27A cells, and treated with etoposide, mitoxantrone or cytarabine for 48 hours before labeling with annexin V for apoptosis assay by FACS. HS-27A cells significantly protected THP-1 and NB-4 cells from all three chemotherapy agents. To discover genes in AML cells that are induced by stromal cells and may contribute to chemotherapy resistance, oligonucleotide microarray analysis was done using total RNA extracted from THP-1 or NB-4 cells cultured alone or co-cultured with HS-27A cells. We identified over 1000 genes that were similarly differentially expressed in both AML cell lines by HS-27A co-culture. Among the significantly upregulated genes was cysteine-rich, angiogenic inducer 61 (CYR61). CYR61 is a secreted, extracellular matrix-associated signaling molecule, which has been related to chemotherapy resistance in breast cancer cells and malignant melanoma cells. To validate the microarray results, qRT-PCR was done with RNA from THP-1 cells and NB-4 cells, cultured alone or co-cultured with HS-27A cells. CYR61 expression was increased over 100-fold in those cell lines when co-cultured with HS-27A cells. Next, we examined CYR61 protein expression by immunocytochemistry, with co-staining for myeloperoxidase to confirm that CYR61-expressing cells were AML cells. This study confirmed that CYR61 was highly expressed in THP-1 cells co-cultured with HS-27A cells, but barely detectable in THP-1 cells cultured alone. Further studies are underway to determine if CYR61 contributes to stroma-induced chemotherapy resistance in AML. This study suggests that CYR61 induced by the microenvironment may promote chemotherapy resistance in AML cells. Elucidating the pathways involved in chemotherapy resistance is likely to result in promising combination therapies to reduce relapse, and thereby improve survival for children with AML. Citation Format: Xin Long, Laszlo Perlaky, Tsz-Kwong Man, Michele S. Redell. Stroma-induced molecules and chemotherapy resistance in acute myeloid leukemia 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 4962. doi:10.1158/1538-7445.AM2013-4962

Tumor Cell-Dependent Differences in Stroma-Induced Changes to Bortezomib Sensitivity.

Abstract 2468In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p<0.02, log-rank tests) after treatment with bortezomib. These observations highlight the notion that tumor cell responses to a given agent in the presence of non-malignant stromal cells can exhibit substantial qualitative and quantitative variation, depending on the specific tumor cell type tested, as well as the particular stromal cell population and conditions of the co-culture. Our findings highlight the need to apply combinatorial high-throughput scalable platforms, such as CS-BLI, to evaluate the different permutations of interactions between tumor cells, non-malignant accessory cells of the microenvironment and administered therapeutics. This study also provides a comprehensive functional oncogenomic framework to identify prognostically relevant molecular mediators of stroma-induced resistance to therapy in MM. Disclosures:Groen:Genmab BV: Research Funding. McMilllin:Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.

CXCR4 Antagonist BKT140 Synergizes with Rituximab, Targeting Non Hodgkin Lymphoma (NHL) In Vitro and In Vivo in a Xenograft Model with Bone Marrow Involvement

Abstract 952 Background:B-cell Non Hodgkin lymphoma (NHL) represents the most common malignant lymphoid neoplasm, with heterogeneous pathophysiological and clinical presentation. It may show systemic, nodal or extra nodal localization in various sites, including bone marrow (BM). Although anti-CD20 antibody rituximab significantly improved the outcome of NHL patients, the relapsed/refractory rates are still high. Chemokine receptor CXCR4 and its ligand CXCL12 are critically involved in the survival and trafficking of normal and malignant B lymphocytes. Taking together with the fact that interaction of malignant B cells with stromal cells via CXCR4/CXCL12 signaling may provide chemo-resistance, we hypothesized that blockade of CXCR4 may antagonize the survival and spreading of lymphoma cells and restore their chemo-sensitivity. Results:The effect of CXCR4 antagonist BKT140 on lymphoma cell growth and rituximab-induced cytotoxicity was tested in vitro and in vivo. Inhibition of CXCR4 with BKT140 in CD20-expressing lymphoma cell lines (BL-2, BJAB, Raji, Ramos, SUDHL4, OCI-Ly7) and primary lymphoma cells from patients with BM involvement resulted in significant inhibition of cell growth and in the induction of cell death, respectively. Combination of BKT140 with rituximab significantly enhanced the cytotoxic effect (apoptosis) against the lymphoma cells in a dose-dependent manner (p<0.01). This effect was concurrent with increase in Annexin V binding, loss of ΔΨm and caspase 3 cleavage. These findings indicate that mitochondrial membrane dysfunction and caspase 3 activation are involved in BKT140- and rituximab-mediated lymphoma cell death. Moreover, we were able to show that rituximab induced CXCR4 expression in lymphoma cell lines and primary lymphoma cells, both on mRNA and cell-surface levels, suggesting the possible interaction between CD20 and CXCR4 pathways in NHL. This interaction provides the rationale for CXCR4 targeting in combination with rituximab treatment. Indeed, highly-expressing CXCR4 BL-2 cells were protected by BM stromal cells from rituximab-induced apoptosis, whereas low-CXCR4 BJAB cells were not. The protective effect of stoma was abrogated by BKT140. To evaluate the in vivo anti-lymphoma effect of BKT140 we established a xenograft model of B-cell lymphoma with BM involvement in mice. Human CXCR4-expressing B NHL cell line, BL-2, was subcutaneously implanted into NOD/SCID mice, resulting in the development of aggressive local tumors which specifically spread to the bone marrow. Following the tumor establishment, mice were injected subcutaneously with BKT140, rituximab or with combination of both. BKT140 inhibited the local tumor progression of BL-2 generated tumors. Furthermore, BKT140 treatment significantly reduced the number of BL-2 tumor cells in the BM by 77% (p<0.01) compared to the untreated mice, while rituximab decreased this number only by 20%. Importantly, the combination treatment of BKT140 with rituximab further decreased the number of viable lymphoma cells in the BM, achieving 93% reduction (p<0.012). We found that BKT140 promoted lymphoma cell apoptosis within the bone marrow microenvironment. Furthermore, we found that the number of viable lymphoma cells in the peripheral blood of tumor-bearing animals was also reduced following the treatment with BKT140. Conclusions:Our results demonstrate potent anti-lymphoma effect of CXCR4-specific antagonist BKT140 in vitro and in vivo. The BKT140-mediated anti-lymphoma effect synergizes with that of Rituximab. Moreover, BKT140 effectively targets lymphoma cells in the bone marrow microenvironment, overcoming the stroma-induced resistance to rituximab. These findings suggest the possible interaction between CD20 and CXCR4 pathways in NHL, and provide the scientific basis for the development of novel combined CXCR4-targeted therapies for refractory NHL. Disclosures:No relevant conflicts of interest to declare.