Absence Of Stromal Cells Research Articles

Pharmacological targeting of netrin-1 inhibits EMT in cancer.

Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFβ1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.

Integrated Functional Genomic and Pharmacological Characterization of the Kinome of Myeloma Cells

Small molecule kinase Inhibitors represent the main therapeutic modality for targeting aberrant kinase signaling in cancer. The recent emergence of CRISPR/Cas9 based functional genomic tools facilitates the genome-wide assessment of cancer cell dependencies, and evaluation of the relative dependence of tumor cells on diverse types of kinases inhibited by small molecule agents with different specificities, as well as other kinases for which selective inhibitors may not be currently available. In this study, we integrated the phenotypic information from the response of multiple myeloma cell lines to small molecule kinase inhibitors (16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure; targeting a total of 43 known primary oncogenic targets) vs. CRISPR-based functional data on the relative dependency of MM lines on the respective targets of these inhibitors. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors, consistent with the high proliferative rates of MM cell lines in vitro and the role of the corresponding genes as broad-spectrum dependencies across not only MM, but also a collection of 300+ cell lines from other neoplasias. We observed virtually no anti-MM in vitro activity for selective inhibitors of c-met, ALK, EGFR, c-kit, PDGFR, VEGFR, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases):, consistent with these results, the corresponding kinases genes were not essential for in vitro survival and proliferation of MM cell lines in our CRISPR studies. We observed modest to minimal response to selective pharmacological inhibitors for PDK1, PI3K (excluding those that also inhibit mTOR), and Akt. Interestingly, at the functional genomic level, PI3KCA was a prominent dependency (and more pronounced than other PI3K isoforms) for several MM cell lines, while individual isoforms of AKT were typically not essential genes for most or all of the MM cell lines tested, suggesting potential redundancy between these isoforms or feedback loops as bypass mechanisms attenuating the effect of pharmacological inhibitors. Prominent and selective MM kinase dependencies at the functional genomic level include PIM2 (providing indirect explanation for the clinical activity observed in Phase 1 trials in MM of a pan-PIM inhibitor) and IKBKB (consistent with the role of the NFKB pathway in MM). Consistent with prior studies on small inhibitors against IGF1R, its gene is a dependency in most MM lines tested and one of the most recurrent dependencies among receptor tyrosine kinases. Both pharmacological and CRISPR-based results suggest heterogeneous responses of MM cells to loss-of-function of MEK1/2 and, more broadly to diverse members of the MAPK family. Comparison of pharmacological results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our results provide novel insights into the relative role of different classes of kinases in MM; the similarities and potential differences in the impact of pharmacological inhibition vs. genomic perturbation of these kinases. These integrated functional genomic and pharmacological studies of the kinome provide a framework for the potential development of inhibitors against previously understudied or underappreciated kinases; and the development of molecular or functional genomic assays that may potentially guide future efforts towards individualized administration of kinase inhibitors in MM. DisclosuresMitsiades:TEVA: Research Funding; Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; Ono: Research Funding; Abbvie: Research Funding; Takeda: Other: Employment of family member.

Long-Term Interactions with Bone Marrow Stromal Cells Alter the Molecular Dependencies of Myeloma Cells: Insights from Genomewide CRISPR-Based Functional Genomic Screens

Bone marrow stromal cells (BMSCs) play important roles in the pathophysiology of multiple myeloma (MM), other hematologic malignancies and metastatic solid tumors, by supporting the survival, proliferation and treatment resistance of malignant cells. Prior molecular profiling studies from our group and others documented that a broad range of proliferative, oncogenic and anti-apoptotic transcripts are upregulated in MM cells by their in vitro coculture with BMSCs in short-term assays (e.g. <1 week of coculture). In this study, we hypothesized that longer-term co-cultures (e.g. over 4+ weeks) with BMSCs are capable of further remodeling of the molecular networks on which MM cells depend for their survival and proliferation.To address this hypothesis, we performed a genome-wide CRISPR/Cas9-based gene editing screen, in which MM.1S cells engineered to stably express the nuclease SpCas9 were also transduced with a lentiviral particles for a pool of ~70,000 sgRNAs (Brunello library), targeting exons of ~20,000 genes (plus non-targeting control sgRNAs), under conditions of transduction which allow for an average of no more than 1 sgRNA to be incorporated in a given cell. This allowed us to convert the initial population of MM.1S cells into heterogeneous pools in which each gene is subject to individual loss-of-function, due to Cas9-mediated editing, by only 1 sgRNA (pool contains typically 4 sgRNAs per gene; 500+ cells per sgRNA; 4 replicates per experimental condition). The MM.1S-Cas9+ Brunello-transduced cells were then cultured in the absence (conventional monoculture) or presence of the immortalized human BMSC lines HS5 or HS27A (transduced with neither Cas9 nor Brunello sgRNAs), which are established BMSCs lines that can increase survival and proliferation of MM and many other tumor cell types in vitro. The monocultures or co-cultures of MM.1S cells were passaged serially over 4 and 6 weeks: each at those time points, MM cells were isolated from the cultures and processed for PCR and next generation sequencing studies to quantify the sgRNAs which exhibited depletion (vs. enrichment) in MM cells with vs. without co-culture with BMSCs.Consistent with our initial hypothesis, we observed that MM.1S cells cocultured for 4 or 6 weeks with each of the 2 BMSC lines exhibited, compared to monocultures of the same duration, statistically significant depletion (at least 3 sgRNAs/gene, MAGECK algorithm) for several genes with known or biologically plausible roles in cell survival or proliferation, including inhibitors of apoptosis, proteasome subunits, ribosomal genes, transcription factors or cofactors, regulators of splicing, autophagy and intracellular trafficking; with higher numbers of significant “hits” after 6 vs. 4 weeks of co-culture with either HS5 or HS27A. Some of these essential genes exhibited sgRNA depletion both in MM cells cultured in the absence of BMSCs and a quantitatively more pronounced sgRNA depletion in co-cultures with BMSCs, suggesting that interaction with BMSCs enhances the functional dependence of MM cells on these genes. Interestingly, some genes (e.g. with roles in translational and transcriptional elongation; mitochondrial organization and metabolism) exhibited preferential depletion of sgRNAs only in the presence, but not absence of BMSCs, suggesting that these genes represent candidate dependencies with preferential role in the context of tumor-stromal interactions. Notably, among the 2500-3000 genes with significant sgRNA depletion in the absence of stromal cells, >200 of genes (including genes involved in RNA and mRNA processing, cell cycle control, epigenetic regulation and cellular response to stress) did not exhibit sgRNA depletion in the presence of BMSCs, suggesting that the BMSC-MM cell interaction may attenuate or abrogate the role of these genes as candidate molecular dependencies in MM.Our study demonstrates the feasibility of applying functional genomic platforms, such CRISPR/Cas9, in the tumor cell compartment of co-culture systems. Our study also documents the ability of stromal co-cultures to remodel the molecular vulnerabilities of MM cells, and supports the notion that many genes play their role as dependencies and potential therapeutic targets preferentially or exclusively in the context of tumor-stromal interactions. DisclosuresMitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding; Takeda: Other: Employment of family member; Abbvie: Research Funding; Ono: Research Funding.

Stromal Cells Promote Neovascular Invasion Across Tissue Interfaces.

Vascular connectivity between adjacent vessel beds within and between tissue compartments is essential to any successful neovascularization process. To establish new connections, growing neovessels must locate other vascular elements during angiogenesis, often crossing matrix and other tissue-associated boundaries and interfaces. How growing neovessels traverse any tissue interface, whether part of the native tissue structure or secondary to a regenerative procedure (e.g., an implant), is not known. In this study, we developed an experimental model of angiogenesis wherein growing neovessels must interact with a 3D interstitial collagen matrix interface that separates two distinct tissue compartments. Using this model, we determined that matrix interfaces act as a barrier to neovessel growth, deflecting growing neovessels parallel to the interface. Computational modeling of the neovessel/matrix biomechanical interactions at the interface demonstrated that differences in collagen fibril density near and at the interface are the likely mechanism of deflection, while fibril alignment guides deflected neovessels along the interface. Interestingly, stromal cells facilitated neovessel interface crossing during angiogenesis via a vascular endothelial growth factor (VEGF)-A dependent process. However, ubiquitous addition of VEGF-A in the absence of stromal cells did not promote interface invasion. Therefore, our findings demonstrate that vascularization of a tissue via angiogenesis involves stromal cells providing positional cues to the growing neovasculature and provides insight into how a microvasculature is organized within a tissue.

Abstract 3037: Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia

Abstract MERTK is a TAM family (TYRO-3, AXL, MERTK) receptor tyrosine kinase that is aberrantly expressed in &amp;gt;80% of primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by the small molecule tyrosine kinase inhibitor (TKI), MRX-2843, induced apoptosis in leukemia cell cultures and prolonged survival in mouse xenograft models of AML, but was not curative. In these models, treatment efficiently reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. To evaluate the role of the bone marrow stromal niche in resistance to MERTK inhibition by MRX-2843, Kasumi-1 and OCI-AML5 AML cell lines were cultured with the fibroblast-like Hs27 cell line and induction of apoptosis and cell death was measured by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with Hs27 cells significantly reduced AML cell death in response to MRX-2843 compared to leukemia cells alone (e.g. Kasumi-1 31.9% vs 61.2%, p&amp;lt;0.05). To identify parallel signaling pathways that may be activated under cytokine rich bone marrow stromal conditions and mediate resistance to MRX-2843, a unbiased phospho-kinase array was performed to identify targets differentially activated by co-culture and/or MRX-2843. Kasumi-1 cells were treated with MRX-2843 in the presence or absence of stromal co-culture, then cell lysates were incubated with human phospho-kinase arrays and proteins were quantitated by densitometry. Five kinases were differentially inhibited by &amp;gt;1.5-fold in the presence or absence of stromal cells, including numerous MAPK pathway components - p38α, ERK1/2, EGFR, TOR, and HSP27. Changes in ERK1/2 phosphorylation (pERK) were confirmed by immunoblot in Kasumi-1 and OCI-AML5 cell cultures. In the absence of co-culture MRX-2843 potently inhibited pERK at doses as low as 100nM, while in co-culture even a 3-fold higher dose did not fully inhibit pERK, consistent with a potential role in resistance. To determine whether the observed upregulation and persistent activation of ERK1/2 has functional significance, Kasumi-1 cells were treated with MRX-2843 and the MEK1/2 TKI pimasertib in the presence or absence of stromal co-culture. Treatment with MRX-2843 or pimasertib alone did not significantly affect survival of AML cells in co-culture (31.3% MRX-2843, 28.1% pimasertib vs 22.7% vehicle, p=ns), but treatment with a combination of MRX-2843 and pimasertib resulted in significant induction of apoptosis compared to vehicle or single TKIs (56.7%, p&amp;lt;0.05). Similar results were observed in OCI-AML5 cell cultures and in combination with the MEK1/2 inhibitor PD0325901. Together these results indicate a critical role for stromal cell-mediated activation of ERK1/2 in resistance to MRX-2843 and demonstrate the utility of translational strategies targeting both MERTK and MAPK pathways to overcome resistance. Citation Format: Katherine A. Minson, Eleana Vasileiadi, Madeline G. Huey, Xiadong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3037.

FoxM1-Mediated Signaling Promotes the Progression of Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL) is an aggressive B-cell lymphoma subtype with elevated B-cell receptor activity. Ibrutinib (IBN), the Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have an overall response rate of 68% in relapsed or refractory MCL patients (Wang et al., NEJM, 2013). However, with the emergence of IBN resistance, novel therapies to thwart resistance are urgently needed. FoxM1 (Forkhead box M1) is a proliferation-associated transcription factor that stimulates cell proliferation and exhibits a proliferation-specific expression pattern. FoxM1 has recently been classified as a human proto-oncogene and we have previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, we investigated the prognostic significance of FoxM1 and its potential as a new MCL therapeutic target.MethodsWe assessed the anti-MCL effects of targeting FoxM1 in both ibrutinib-sensitive and -resistant MCL cell lines and clinical specimens. Cell viability assays were conducted targeting FoxM1 with thiostrepton, a published FoxM1 inhibitor. The drug screening was performed in a 96-well format in which MCL cells were seeded at 10,000 cells per well and were treated with the FoxM1 inhibitor thiostrepton at the following concentrations: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 uM. Cell viability was tested using the CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation period. Furthermore, we investigated the importance of FoxM1 signaling in tumor migration and adhesion in Jeko-1 WT and BTK KD (generated from Jeko-1 using CRISPR/Cas9) cells. Jeko-1 WT and two BTK KD variants were transiently transfected with control siRNA and siRNA against FoxM1 (siFoxM1) for 24 and 48 hours. Afterwards, cells were loaded into transwell migration inserts in the presence or absence of human stromal cells. Migration was evaluated by counting migrated cells and normalizing the migrated cells to migration in the absence of stromal cells. The in vivo efficacy of thiostrepton was evaluated in two cell line Jeko-1 and Jeko-1 BTK KD mouse xenograft models. Upon engraftment, thiostrepton was administered intravenously five consecutive days a week at 50 mg/kg, and tumor burden was assessed via the measurement of circulating human β2M levels and tumor volume. FoxM1 plasma levels in the xenografted mice were also evaluated using ELISA at 0, 10, 20, and 30 days.ResultsInhibition of FoxM1 with thiostrepton reduced the cell viability of both ibrutinib-sensitive (Jeko-1; SP-49; PT-1; PT-2) cell lines and patient samples as well as ibrutinib-resistant MCL cells (Maver-1; Z138; Jeko-BTK KD 1 and 2; PTs 3-6) at half-maximal inhibitory concentration (IC50) in the micromolar range (IC50 = 1-3 μM) for the majority of tested cells and patient samples. siFoxM1 significantly reduced (P < 0.05) stromal cell-mediated migration of both Jeko-1 WT and BTK KD cells compared with the cells transfected with control siRNA. Moreover, siFoxM1-treated cells showed reduced levels of the migration- and adhesion-related proteins snail, vimentin, and N-cadherin compared with the control cells. Additionally, in comparison to vehicle-treated control mice, thiostrepton treatment significantly reduced tumor volume (36% and 17%, respectively) and β2M levels (71% and 79%, respectively) in Jeko-1 and Jeko-1 BTK KO xenografted mice at Day 30 of treatment regardless of BTK status. Lastly, thiostrepton treatment significantly suppressed the plasma levels of FoxM1 in both Jeko-1 (14%) and Jeko-1 BTK KO (11%) xenografted mice at Day 30 of treatment.ConclusionWe have shown that FoxM1 inhibition may be a potential candidate treatment for MCL based on the results of our clinicopathological assessment and in vivo studies. Therefore, exploring the role of FoxM1 in MCL disease progression and therapeutic resistance may lead to novel therapeutic breakthroughs to improve patient clinical outcomes. DisclosuresWang:MoreHealth: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Research Funding; Kite Pharma: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Honoraria, Research Funding; Juno: Research Funding; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria.

Abstract A066: NPRA signaling in the tumor/stroma microenvironment influences the growth of stem-like cancer cells

Abstract Alterations to NPRA signaling may lead to inhibition of cancer stem cell (CSC) proliferation; we report the development of a 3D tumor/stroma coculture model to study and further define the role that NPRA plays in CSC development and drug resistance. Objectives: Determine changes in cell phenotype caused by culture on a 3D nanofiber scaffold or suspension culture, whether these changes represent an enrichment of CSCs, the mechanism of enrichment/role of NPRA. Examine the roles of stromal cell types in tumor growth, CSC development, and drug resistance in an in vitro coculture. Assess CSC expansion and drug resistance in 3D scaffold tumoroids or suspension with/without cancer associated fibroblasts (CAF) as well as in isolated CSC populations. Methods: CSCs were assayed by flow cytometry (CD44+/CD24-, CD133+) and isolated by FACS or MACS. Stemness was assayed by SOX2 OCT4 NANOG expression and aldehyde dehydrogenase(ALDH) activity with the ALDEFLUOR kit. Sphere forming assays (SFAs) for anchorage independent growth with/without NPRA inhibitors and/or stromal cells were performed and measured by bright field microscopy. Tumor initiation was assayed in the flanks of syngeneic or NSG mice. NPRA KO mice were used to determine the influence of stromal NPRA on tumor growth. Cancer cells were grown on 3D nanofiber scaffold with/without stromal cells as an in vitro model of the tumor microenvironment. CSC enrichment was assayed by CSC marker expression and SFAs. NPRA knockout cell lines were made using CRISPR/Cas9, verified by flow cytometry and qPCR. Knockout cell lines were tested for their tumor initiation, CSC marker expression, and SFA in the presence or absence of stromal cells. Results: Introduction of factors secreted by CAF increased the tumor cells' ability for attachment-independent growth; however, this was not seen with NPRA inhibitor treatment. Putative CSCs collected from tumor spheres initiated tumors in mice at lower cell numbers than their parental cell lines. 3D culture of tumor biopsies increased expression of CD44+ CD24- ALDH+ compared to primary tumor or monolayer. 3D culture increased the drug resistance of cancer cells, compared to 2D. Cells from the CD44+CD24- population initiated tumors at lower numbers than parental. Tumor spheres initiated in vivo tumors at lower cell counts than monolayer cultures. These tumors grew faster in wild type mice vs NPRA KO mice. CRISPR genome editing allowed us to create NPRA knockout cell lines to confirm the results of the inhibitor studies. Coculture of different cell types with/without NPRA KO determined NPRA's influence on CAF supporting tumor growth. Conclusions: The 3D scaffold offers a cell culture environment causing cancer cells to retain more in vivo behavior. This model can increase the CSC phenotype and can be used to study the mechanisms of CSC proliferation, drug resistance, and the role of stromal cells. We hypothesized that NPRA signaling is key for this process and we found that inhibiting NPRA signaling reduces the ability for attachment-independent growth even in the presence of tumor supporting CAF and their in vivo tumor initiation ability. Cancer cell growth was reduced by NPRA inhibition and NPRA signaling is important for the initiation and maintenance of tumors. We plan to use the scaffold model to determine NPRA signaling’s influences on CSC development and its effects on each stromal cell type within tumors. Citation Format: Ryan J. Green, Mark Howell, Rajesh Nair, Shruti Padhee, Scott Antonia, Eva Samal, Jit Banerjee, Shyam Mohapatra, Subhra Mohapatra. NPRA signaling in the tumor/stroma microenvironment influences the growth of stem-like cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A066.

Targeting the CXCR4 pathway using a novel anti-CXCR4 IgG1 antibody (PF-06747143) in chronic lymphocytic leukemia

BackgroundThe CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL.MethodsPatient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model.ResultsPF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine.ConclusionsWe show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.

Ig Light Chain Precedes Heavy Chain Gene Rearrangement during Development of B Cells in Swine.

The current mammalian paradigm states that 1) rearrangements in the IgH locus precede those in IgL loci, 2) IgLλ genes rearrange only when IgLκ genes are consumed, and 3) the surrogate L chain is necessary for selection of productive IgH gene rearrangements. We show in swine that IgL rearrangements precede IgH gene rearrangements, resulting in the expression of naked IgL on a surface of precursor B cells. Findings also suggest that there is no dependency on the surrogate L chain, and thus the authentic IgL proteins may be used for selection of the IgH repertoire. Although rearrangement starts with IgLκ genes, it is rapidly replaced by IgLλ rearrangement. Fast replacement is characterized by occurrence of IgLλloIgLκlo dual-expressing precursors in which IgLκ expression is a remnant of a previous translation. Most IgLκ+ B cells are then generated later, indicating that there are two waves of IgLκ synthesis in different developmental stages with IgLλ gene rearrangements in between. In the absence of stromal cells, the stepwise order of rearrangements is blocked so that IgLλ gene rearrangements predominate in early B cell development. To our knowledge, this is the first evidence that some mammals can use an inverted order of Ig loci rearrangement. Moreover, a situation in which the generation of BCR-bearing IgLκ is delayed until after IgLλ becomes the dominant isotype may help explain the extreme deviations in the IgLκ/IgLλ ratios among mammals.

Growth of human breast tissues from patient cells in 3D hydrogel scaffolds

BackgroundThree-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells.MethodsPrimary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer.ResultsWhen seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix.ConclusionsThese 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0677-5) contains supplementary material, which is available to authorized users.

The Combination of the Novel Syk Inhibitor TAK659 with Decitabine Exerts Synergistic Cytotoxic Effects Against FLT3/ITD Mutated Acute Myeloid Leukemia Cells

Background: Effective treatment regimens for FLT3/ITD+ AML patients are lacking. Recent data on the effects of FLT3 tyrosine kinase inhibition showed promising clinical activity, but outcomes have been stunted by drug resistance. Targeting additional signaling pathways besides FLT3 might therefore offer therapeutic benefit. Spleen tyrosine kinase (syk) is frequently overexpressed and constitutively activated in primary AML FLT3/ITD+ blasts. Pharmacologic inhibition of syk has been associated with antileukemic potential, consisting of inhibition of cell growth and induction of differentiation in AML cells. Hypomethylating agents (DNMTis) such as Decitabine (DEC) have emerged as attractive agents in the treatment of AML, partially due to their proapoptotic and differentiation inducing effects. There is a strong rationale for combining FLT3- and syk- inhibitors with DNMTis for FLT3/ITD+ AML. Here, we investigated whether the antileukemic effects of the novel, dual FLT3/syk inhibitor TAK659 (TAK) can be enhanced by the co-administration of DEC.Methods: Molm14 (M14) cells were incubated in culture medium in the presence and absence of stromal cells (MS5) for 72 hours. TAK or DEC was added in single agent or combined mode at the indicated concentrations. After the incubation period, cell growth was determined by MTT assays. Apoptosis, autophagy and cell cycle status were assessed by FACS analysis. FLT3- and syk- phosphorylation levels were detected by western blotting. Differentiation was assessed by morphology. A combination index (CI) was calculated using Calcusyn Software.Results: TAK exerted dose dependent effects against M14 cells. However, the presence of stromal cells significantly blunted TAK mediated cytotoxicity (50nM: 61.9±1.3% [off stroma] vs. 10.9±3.1% [on stroma], p<.001; 500nM: 91.2±0.5% [off stroma] vs. 17.4±2.5% [on stroma], p<.001, n=3-9; IC50: 40.6nM [off stroma] vs. 3.9µM [on stroma]). Although less marked when stroma was present, TAK combined with DEC yielded synergistically enhanced growth inhibition (off stroma: 50.2±3.9% [TAK659 50nM], 58.1±3.0% [DEC 1µM], 76.7±3.1% [TAK659 50nM+DEC 1µM; CI=0.77]; on stroma: 9.5±3.0%[TAK659 50nM], 42.9±3.1% [DEC1µM], 57.0±5.2%[TAK659 50nM+DEC 1µM; CI=0.62]) compared to single agent treatment (off stroma: p<.001 vs. TAK, p< .001 vs. DEC, on stroma: p<.001 vs.TAK, p=.03 vs. DEC, n=3). In line with these findings, combined treatment yielded greater levels of FLT3- and syk- dephosphorylation compared to single agent treatment both when stroma was present and absent. When compared to untreated controls (11.6±0.7%), Annexin V/PI staining (n=3) showed that induction of apoptosis was increased with TAK (18.9±3.9%; p=NS), DEC (39.2±4.8%; p<.01), and greatest when the two drugs were combined (44.2±6.7%; p<.01) in the absence stoma. In the presence of stroma, single agent DEC, and when combined with TAK, increased the fraction of apoptotic cells by up to two fold whereas TAK did not induce any meaningful apoptosis. Consistent with the induction of cell death, cell cycle analysis demonstrated the greatest increase in subG0 phase when TAK and DEC were combined. Both in the presence and absence of stroma, autophagic flux was increased when M14 cells were treated with DEC, and when DEC was combined with TAK whereas no difference was observed with TAK alone. The combination of TAK and DEC led to changes consistent with differentiation, as shown by more abundant cytoplasm and nuclei with deep indentations, folds and lobulation compared to untreated cells. DEC treated cells also showed signs of differentiation although these changes were less marked. No evidence of differentiation was observed in M14 cells when treated with TAK alone.Conclusions: 1. Stroma conferred protective effects against all treatments. 2. TAK exerted dose dependent cytotoxicity against M14 cells in the nanomolar to low micromolar range in the absence and presence of stroma, respectively. 3. The cytotoxic effects of TAK were enhanced when TAK was combined with DEC. 4. The antileukemic effects of TAK combined with DEC are multifaceted and include inhibition of cell growth, induction of apoptosis, autophagy and differentiation. 5. The combination of TAK and DEC represents a potentially important novel approach for FLT3/ITD+ AML patients. 6. Clinical trials incorporating syk-inhibitors into standard AML treatment protocols are currently under way. DisclosuresDos Santos:Amgen: Employment.

Constitutive Vs. Stroma-Induced Kinase Dependencies in Myeloma Cells: Functional Mapping Using Small Molecule Inhibitors As Chemical Probes

The functional characterization of kinases in multiple myeloma (MM) cells has mainly involved the use of RNAi, a mechanistic approach inherently dissimilar to small molecule inhibitors that are applied in the clinic. Furthermore, in the bone marrow (BM) microenvironment, where MM cells primarily reside, BM stromal cells (BMSCs) and other nonmalignant cell populations can function as "accessory" cells and alter the vulnerability of MM cells to diverse targeted therapies, including kinase inhibitors. In order to characterize how the kinase dependencies of MM cells differ in the presence vs. absence of BMSCs, we evaluated 16 MM cell lines and their response, in monoculture vs. co-culture conditions, to a panel of 273 kinase inhibitors (100nM, 24-72 h exposure), which target a total of 43 known primary oncogenic targets. We observed that virtually all tested cell lines showed no response, in either monocultures or co-cultures, to inhibitors of c-met, ALK, Abl, EGFR superfamily members (EGFR, HER2), c-kit, PDGFR, Flt3, FAK, VEGFR and Syk, including cell lines with detectable transcript levels for the respective kinases at baseline or stroma-induced increase in co-cultures. These results suggest that the respective kinases do not represent major dependencies for MM cell lines in either the cell-autonomous state or in the context of interaction with stromal cells. Dual PI3K/mTOR inhibitors exhibited similarly potent anti-tumor activity in both monocultures and stromal co-cultures of essentially all MM cell lines tested; while mTOR inhibitors devoid of PI3K activity had examples (e.g. in U266 and KMM2 cells) of stroma-induced resistance, likely reflecting a role of PI3K signaling in mediating, at least in some MM cells, stroma-induced decrease in dependence to mTOR function. Inhibitors of IKK or JAK (e.g. ruxolitinib, baricitinib) exhibited higher activity against several MM cell lines in the presence compared to absence of BMSCs. This stroma-induced sensitization to these kinase inhibitors is consistent with the role of BMSC-derived cytokines or cell adhesion molecules in inducing in MM cells NF-kappaB transcriptional activation or JAK/STAT3 signaling. Interestingly, several other classes of kinase inhibitors, e.g. against Aurora, PLK, MEK, Akt, exhibited more heterogeneous stroma-induced effects on their activity (decrease against some cell lines vs. sensitization against others; without an obvious common pattern of these effects for each of these kinase inhibitors across different cell lines). These results suggest that the dependence of MM cells on the corresponding kinases and the signaling networks they regulate are not only subject to stroma-induced changes, but also exhibit a higher degree of cell type-dependent plasticity than previously appreciated. In further support of the complexity of these stoma-induced events, we also observed that BRAF inhibitors (e.g. vemurafenib, dabrafenib, PLX-4720, AZ-628, et.c.) induced at least modest increase in proliferation of several BRAF-wild-type MM cell lines cultured in the absence of stromal cells, but co-culture with BMSC blunted this effect in some cell lines (JJN3, AMO1, OPM2), and modestly enhanced it in others (e.g. KMS34). BRAF inhibitor-induced proliferation of BRAF-wild-type cell lines has been previously reported in different tumor types and has been attributed to activation and signaling through C-RAF: our current observations suggest that, in MM patients harboring both V600E-BRAF mutant and wild-type clones, the impact of treatment with BRAF inhibitor on decreasing the burden of the former clone(s) vs. selecting for outgrowth of the latter may actually be subject to complex, genotype-dependent, influence of the BMSCs of the local microenvironment. In summary, our studies demonstrate the feasibility of functionally annotating the kinase dependencies in MM and potentially other neoplasias by using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of tumor cell lines. Furthermore, our studies suggest that future efforts to individualize the administration of kinase inhibitors in MM should take into account not only the genotype of MM cells in the respective patient, but also the heterogeneous impact that nonmalignant "accessory" cells such as BMSCs can have on the MM cell dependence on each respective kinase and its downstream targets. DisclosuresAftab:Cleave Biosciences, Inc.: Research Funding; Onyx Pharmaceuticals, Inc.: Research Funding; Atara Biotherapeutics, Inc.: Employment, Equity Ownership; Omniox, Inc.: Research Funding. Mitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding.

Induction of differentiation of human embryonic stem cells into functional hair-cell-like cells in the absence of stromal cells

Sensorineural hearing loss and vestibular dysfunction have become the most common forms of sensory defects. Stem cell-based therapeutic strategies for curing hearing loss are being developed. Several attempts to develop hair cells by using chicken utricle stromal cells as feeder cells have resulted in phenotypic conversion of stem cells into inner ear hair-cell-like cells. Here, we induced the differentiation of human embryonic stem cells (hESCs) into otic epithelial progenitors (OEPs), and further induced the differentiation of OEPs into hair-cell-like cells using different substrates. Our results showed that OEPs cultured on the chicken utricle stromal cells with the induction medium could differentiate into hair-cell-like cells with stereociliary bundles. Co-culture with stromal cells, however, may be problematic for subsequent examination of the induced hair-cell-like cells. In order to avoid the interference from stromal cells, we cultured OEPs on laminin with different induction media and examined the effects of the induction medium on the differentiation potentials of OEPs into hair-cell-like cells. The results revealed that the culture of OEPs on laminin with the conditioned medium from chicken utricle stromal cells supplemented with EGF and all-trans retinoic acid (RA) could promote the organization of cells into epithelial clusters displaying hair-cell-like cells with stereociliary bundles. These cells also displayed the expected electrophysiological properties.

Functional mapping of multiple myeloma kinome with library of small molecule inhibitors

The functional role of individual kinases in multiple myeloma (MM) cells has been primarily interrogated using RNAi approaches, which are mechanistically dissimilar to the pharmacological action of small molecule inhibitors used in the clinic; and in stroma-free cultures, which don’t account for stroma-induced signaling events. To address these dissimilarities, we performed functional mapping of the kinome in 16 MM cell lines, cultured in the presence vs. absence of stromal cells, using a panel of 273 kinase inhibitors (100nM, 24-72 h exposure) targeting a total of 43 known primary oncogenic targets. The cell-autonomous activity of the various inhibitors was broadly potent for Aurora, PLK, and mTORC1/2 inhibitors (consistent with high proliferative rates of MM cell lines in vitro); modest to minimal for selective PDK1, PI3K (excluding those that also inhibit mTOR), and Akt inhibitors; virtually none for selective inhibitors of c-met, ALK, EGFR, HER2, c-kit, PDGFR, VEGFR, Flt3, FAK, Syk, Src, and BTK (even in cell lines with detectable transcripts for the respective kinases). We also observed modest activity for FGFR3 inhibitors against FGFR3expressing cell lines; and none for BRAF inhibitors in our BRAF wild-type cell lines (some of which had increased proliferation upon treatment; consistent with known adaptive activation of signaling through C-RAF). Testing 8 different MEK1/2 inhibitors identified lines with low, intermediate vs. high response; the latter being KRAS-mutant and BRAF-wild-type cells with high baseline p-ERK levels (e.g. due to MEK2-activating mutation in one cell line). Importantly, comparison of results obtained in the presence vs. absence of stroma, indicate heterogeneous effect of bone marrow stromal cells on MM cell response to inhibitors across different targets and cell lines, including stroma-induced resistance to the same kinase inhibitor for some cell lines vs. sensitization for others. Our studies demonstrate the feasibility of functional annotations of the kinome in MM using libraries of small-molecule kinase inhibitors in phenotypic assays against panels of cell lines. Our results provide insight into the relative role of different classes of kinases in MM, and how the genetic (e.g. MEK2 or BRAF mutation status) and microenvironmental context can influence the role of these kinases and possible future individualized administration of inhibitors against these kinases in MM. BP-032

Temporal Dynamics of Tumor-Microenvironment Interaction and Treatment Responses Revealed through Time-Lapse Compartment-Specific Bioluminescence Imaging: Translational implications

Most conventional methods to sensitively quantify tumor cell proliferation and viability in vitro involve processing of cells in ways that preclude continuation of the respective experiment or prevent the longitudinal collection of data. This common technical feature of conventional assays limits their ability to provide detailed insight into the kinetics of tumor cell responses to treatment(s). Additionally, these limitations hinder the use of these assays to monitor how the kinetics of treatment response can be altered by nonmalignant “accessory” cells of the tumor microenvironment (e.g. bone marrow stromal cells [BMSCs] for hematologic malignancies or bone metastases of solid tumors). To address these obstacles, we modified our previously developed tumor cell compartment-specific bioluminescence imaging (CS-BLI) platform (McMillin et al. Nat Med. 2010), to enable longitudinal assessment of tumor cell response to diverse experimental conditions; we cultured luciferase-expressing tumor cells, with or without stromal cells, in the presence of bioluminescent substrates, using optimized conditions which provide detectable bioluminescent signal even after several days of culture, while having no adverse effect on the viability of tumor or non-malignant cells in this system. This modified approach (time-lapse CSBLI, [TL-CSBLI]) preserved the linear correlation of bioluminescent signal with tumor cell viability. Furthermore, results obtained at the end of the experiment and during interim time-points are consistent with those generated using either non-time-lapse applications of CS-BLI or conventional techniques. We applied TL-CSBLI to delineate, in high-throughput manner, the temporal dynamics of the responses of tumor cells (e.g. multiple myeloma (MM) and other hematologic malignancies) to diverse treatments (e.g. conventional chemotherapeutics, glucocorticoids; proteasome inhibitors (PIs, bortezomib or carfilzomib), and kinase inhibitors). Using the time-lapse capabilities of this assay, we evaluated tumor cell responses in the presence vs. absence of stromal cells. We observed that the kinetics of tumor cell response to diverse therapeutic classes are heterogeneous, even within the same tumor type: for instance, tumor cells with pronounced responses at the end of drug incubation (e.g. 24, 48, 72, hrs after initiation of treatment with PIs, DNA-damaging chemotherapeutics, or dexamethasone respectively), can have different magnitude of responses at intermediate time points. This suggests that TL-CSBLI data can further stratify treatment-responsive tumor cells into those with early vs. late kinetics of response. We also observed that the kinetics of the proliferative / anti-apoptotic effect conferred by stromal cells on tumor cells are highly variable between different cell lines, even within the same tumor type. For instance, the time between initiation of coculture and maximum stimulation of tumor cell viability by stromal cells was variable between cell lines and did not correlate with the magnitude of stimulation by stromal cells. Importantly, TL-CSBLI identified that the response of diverse types of tumor cells to treatments can be delayed in the presence of stromal cells, compared to conventional tumor cell monocultures: this initial delay in treatment response of tumor cells in stromal co-cultures may be observed even in cases where similar cytoreductive responses are eventually observed at later time-points in both the presence and absence of stromal cells. This observation suggests that a more expansive definition of stroma-induced resistance to a given treatment may be warranted, to specifically incorporate the ability of stromal cells to delay the tumor cell response to such treatment. In summary, TL-CSBLI enables detailed characterization of the kinetics of tumor cell responses to diverse experimental conditions. Its use can provide insight into the underappreciated impact that cell-autonomous variations or stroma-induced changes in the kinetics of tumor cell response to a given anti-tumor therapy can have on determining its efficacy. This is particularly consequential for agents (e.g. PIs) which have clinical pharmacokinetic profiles associated with transient peak exposure. DisclosuresMcMillin:Axios Biosciences: Equity Ownership; DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Negri:DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.

Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells.

The efficacy of donor HSCT is partly reduced as a result of slow post-transplantation immune recovery. In particular, T cell regeneration is generally delayed, resulting in high infection-related mortality in the first years post-transplantation. Adoptive transfer of in vitro-generated human T cell progenitors seems a promising approach to accelerate T cell recovery in immunocompromised patients. AA may enhance T cell proliferation and differentiation in a controlled, feeder-free environment containing Notch ligands and defined growth factors. Our experiments show a pivotal role for AA during human in vitro T cell development. The blocking of NOS diminished this effect, indicating a role for the citrulline/NO cycle. AA promotes the transition of proT1 to proT2 cells and of preT to DP T cells. Furthermore, the addition of AA to feeder cocultures resulted in development of DP and SP T cells, whereas without AA, a preT cell-stage arrest occurred. We conclude that neither DLL4-expressing feeder cells nor feeder cell conditioned media are required for generating DP T cells from CB and G-CSF-mobilized HSCs and that generation and proliferation of proT and DP T cells are greatly improved by AA. This technology could potentially be used to generate T cell progenitors for adoptive therapy.

A Method for Measurement of Drug Sensitivity of Myeloma Cells Co-Cultured with Bone Marrow Stromal Cells

The tumor microenvironment can profoundly affect tumor cell survival as well as alter antitumor drug activity. However, conventional anticancer drug screening typically is performed in the absence of stromal cells. Here, we analyzed survival of myeloma cells co-cultured with bone marrow stromal cells (BMSC) using an automated fluorescence microscope platform, ScanR. By staining the cell nuclei with DRAQ5, we could distinguish between BMSC and myeloma cells, based on their staining intensity and nuclear shape. Using the apoptotic marker YO-PRO-1, the effects of drug treatment on the viability of the myeloma cells in the presence of stromal cells could be measured. The method does not require cell staining before incubation with drugs, and less than 5000 cells are required per condition. The method can be used for large-scale screening of anticancer drugs on primary myeloma cells. This study shows the importance of stromal cell support for primary myeloma cell survival in vitro, as half of the cell samples had a marked increase in their viability when cultured in the presence of BMSC. Stromal cell–induced protection against common myeloma drugs is also observed with this method.

Beta7 Integrins Regulate Podia Formation in Multiple Myeloma (MM) Cells for the Interaction with the Cellular and Non-Cellular Bone Marrow (BM) Stroma

AbstractAbstract 3979Adhesion of MM cells to the BM microenvironment can lead to cell adhesion mediated drug resistance (CAM-DR). We have previously shown that treatment with Dexamethasone (a drug commonly used against MM alone or in combination with new therapeutic agents) induces in MM cells increased assembly of CD138, and F-actin containing membrane extensions named podia. Using this 2D co-culture model we found that podia from MM cells from patients or MM cell lines elongate on the surface of BM stromal cells and also interconnect distant MM cells correlating with CAM-DR. Formation of podia was observed after at least 6 days of co-culture when many soluble factors and extracellular matrix proteins may have been secreted and remodelled. We also showed that preventing SDF1-alpha binding to the CXCR4 receptor in MM cells inhibits podia formation. In order to investigate the possible role of ECM proteins and their ligands in podia assembly we have now tested the formation of podia in 3D co-cultures of MM and stromal cells embedded in gels composed of polymerised extracellular matrix proteins found in the BM including collagen I and collagen IV. We now found that MM cells incubated overnight in 3D matrix lattices assembled podia that elongated along fibres of polymerised extracellular matrix that put them in contact with stromal cells or other distant MM cells. We also found that culture of MM cells in 3D matrices in the absence of stromal cells did not result in podia formation. These results suggest that stromal cells promote podia formation in contact with the extracellular matrix. It has been shown by Neri et al1that beta7 integrins are involved in MM cell adhesion to BM cells and extracellular matrix during recruitment to the BM and they are involved in CAM-DR. We found that integrin beta7 KD MM cells failed to form podia to the same extent as the parental cell lines in 2D and 3D cultures. Podia assembled by beta7 KD cells were shorter and highly irregular in shape in comparison to long and straight podia formed by parental MM cells. Using immunostaining, we found that podia in beta7 KD cells lacked internal F-actin as well as vimentin filaments that we identified in podia of parental MM cells. However, total levels of these cytoskeletal proteins remained unchanged as detected by western blot. Dexamethasone-induced increased formation of podia was also dependent on the expression of beta7 integrins by MM cells and the presence of BM stromal cells in 2D and 3D culture conditions.We conclude that podia promote the formation of cellular networks of distant MM cells as well as the contact with BM stromal cells in 3D and this process is involved in CAM-DR against Dexamethasone. Podia assemble in contact with stromal cells and along extracellular matrix cables and require polymerisation of actin and vimentin cytoskeletal filaments downstream of beta7 integrins. Disclosures:No relevant conflicts of interest to declare.

Bortezomib Can Overcome Stroma-Mediated Chemotherapy Resistance in Acute Lymphoblastic Leukemia.

Abstract 2475 Background:In spite of excellent results of chemotherapy induction resulting in high rates of complete remission in patients with ALL, relapse remains a major problem. This is likely due to a subset of leukemic cells that are chemotherapy resistant. The interaction between bone marrow stromal cells and leukemia cells protects leukemic cells from the cytotoxic effects of chemotherapy. Bortezomib is a proteozome inhibitor used to treat lymphoid malignancies. It has been shown to mobilize stem cells in myeloma patients. We hypothesized that, in addition to its direct cytotoxic effect, bortezomib can also overcome the protective effect of stromal cells by disrupting CXCR4 signaling through inhibition of CXCR4 turnover normally regulated by proteosome degradation. Methods:We used the human B-cell ALL cell lines, G2 and BV-173, and two human BM stromal cell lines, HS-5 and HS-27a for our studies. G2 cells were incubated alone or co-cultured with HS-5 or HS-27a for 24 hours then treated with RPMI (control), cytarabine, doxorubicin, or bortezomib. After 48 hours, cells were harvested and stained with FITC-conjugated anti-human annexin V antibody and analyzed by flow cytometry. To test the effect of bortezomib on CXCR4 expression, G2 cells were incubated with or without stromal cells and treated with cytarabine or bortezomib for 18 hours or left untreated. Then cells were stained with PE-conjugated anti-human CXCR4 antibody (clone 1D9) and analyzed by flow cyometry. The CXCR4 expression was reported as relative mean fluorescent intensity (RMFI) compared to isotype control. We then performed cell cycle analysis of G2 cells treated with cytarabine or bortezomib for 18 hours. All cells were treated with BrdU 1 hour prior to harvest, then stained with APC-conjugated anti-BrdU antibody and analyzed by flow cytometry. Results:Incubation of G2 cells with HS-5 protected them from apoptosis induced by cytarabine or doxorubicin but not bortezomib. The apoptosis rate for cytarabine treated cells was 55±0.3% without HS-5 vs. 15%±0.8 with HS-5(p<0.0001). The apoptosis rate of doxorubicin treated cells was 23±0.6% without HS-5 vs. 4.2±0.8% with HS-5 (p<0.0001). In the case of bortezomib, the apoptosis rate was 67±0.6% without HS-5 vs. 71±1.2% with HS-5. Similar results were observed with the BV-173 ALL cell line and with the use of HS-27a stromal cell line. We noted down-regulation of CXCR4 expression when G2 cells were co-cultured with stromal cells or after bortezomib treatment. In the absence of stromal cells, the CXCR4 RMFI was 40±3 in untreated cells vs. 16±0.4 in bortezomib treated cells (P=0.0015). In the presence of stromal cells CXCR4 RMFI 15±0.7% in untreated cells vs. 5±0.2% in bortezomib treated cells (P=0.0002). In cell cycle analysis, we found only 1.03±0.2% of cells treated with cytarabine were in S phase compared to 10.05±0.7% when cells were treated with bortezomib (P=0.0003). Conclusion:Bortezomib can overcome the stromal cell-mediated protection of human G2 ALL cells. This effect may be, in part, mediated by in the decreased surface expression of CXCR4 shown by others to mediate leukemia-stroma interaction and chemoprotection. In contrast to cytarabine which selectively kills cells in S phase, bortezomib induced killing of ALL cells regardless of cell cycle status. Future studies will use of in vivo mouse models of ALL to test the effects of bortezomib alone or in combination with chemotherapy on survival. Disclosures:DiPersio:genzyme: Honoraria.

Abstract 1511: CXCR4 inhibition sensitizes prostate cancer cells to radiotherapy in an in vitro co-culture model

Abstract Background: Tumor microenvironment is an important factor influencing radiosensitivity of cancer cells. Amongst multiple soluble factors released by tumor-associated stromal cells, chemokine CXCL12 was shown to confer pro-survival effects on cancer cells expressing chemokine receptor 4 (CXCR4). It has been recently shown that stromal microenvironment protects prostate cancer cells from chemotherapy-induced cell death via CXCR4 signaling, which was reversed by AMD3100, a specific CXCR4 inhibitor. In this study, AMD3100 was tested for its radiosensitizing capacity in prostate cancer. Methods: Both short-term (72 h) and long-term survival (17 days) of PC3 prostate cancer cells in the absence or presence of murine stromal cells (MS5) after increasing doses of gamma-irradiation (0-15 Gy) was assessed. To analyze the short-term effect of stromal cells on PC3 viability after radiation, fluorescently labeled PC3 cells were cultured with or without MS5 cells and exposed to gamma-irradiation (0-15 Gy) in the presence or absence of 25 µg/ml AMD3100. DAPI nuclear staining was used to assess the viability of PC3 after 72 hours, Long-term survival of PC3 cells was evaluated by clonogenic assay of PC3 cells seeded alone or on feeder layer of MS5-GFP stromal cells and irradiated with 0-15 Gy. Results: The short-term viability assay showed that PC3 in mono-culture show dose-dependent loss of viability after increasing doses of irradiation, showing only 18±6% (mean ± SD) viability when exposed to 15 Gy as compared to non-irradiated control cells. However, in the presence of stromal cells, viability of PC3 cells exposed to the same radiation dose remained high, reaching levels of 69±2% viability (P=0.004 vs 15 Gy in monoculture). This effect was potently reversed by AMD3100, which lowered viability of cells to 46±3% (P=0.001 vs 15 Gy in co-culture without AMD3100). Importantly, addition of AMD3100 to PC3 cultured in the absence of stromal cells did not change their viability after radiation. The long-term survival measured in clonogenic assay showed that in presence of MS5-GFP murine stromal cell line, 8% PC3-luc cells survived 6 Gy gamma-radiation dose, as compared to only 3.6% of PC3 cells that were cultured without the stromal cells (P=0.03). Conclusion: Murine stromal cells protect PC3 prostate cancer cells against irradiation-induced cell death in an in vitro co-culture system. Treatment with AMD3100 potently inhibits the protective effect of stromal cells on the prostate cancer cells exposed to gamma-irradiation. These data underscore the relevance of stromal microenvironment in tumor response to irradiation and warrant further research on the role of CXCR4 inhibitors as a therapeutic option to potentiate radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1511. doi:1538-7445.AM2012-1511