HS-5 Bone Marrow Stromal Cells Research Articles

Stroma-Based Versus Stroma-Free Long-Term Survival and Proliferation of Primary Acute Myeloid Leukemia (AML) Cells

Background: Acute myeloid leukemia (AML) is a very aggressive hematological malignancy that carries an overall poor prognosis. High throughput screening of primary (patient-derived) leukemic cells is becoming increasingly valuable as a preclinical tool to help individualize the treatment of relapsed/refractory AML patients. However, the necessity of robust in-vitro culture conditions has often limited the optimization of high throughput drug sensitivity testing. Our lab has previously validated a high throughput assay in 384-well format that utilizes primary leukemic cells maintained in a cocktail of cytokines tested for chemosensitivity and resistance against anti-leukemic agents (Shum et al. Clinical Lymphoma, Myeloma and Leukemia 2013). However, cytokines have not been shown to support long-term proliferation of primary AML cells; nor does it assess drug candidates within the appropriate tumor microenvironment. Stromal cells of the bone marrow microenvironment, however, have been reported to support not only the growth and maintenance of normal hematopoiesis, but also the long-term, in-vitro growth of leukemic cells, while also influencing their response to chemotherapy agents. A comprehensive comparison of primary AML cell growth under our validated cytokine assay and stromal conditions has not previously been undertaken. In this study, we compared the long-term proliferation and phenotypes of primary AML cells in high-throughput format under different stromal conditions against cytokines only. Results from this study would help optimize parameters for a high-throughput drug sensitivity testing comparing the differential response to anti-leukemic agents under cytokine versus stromal conditions.Methods: The proliferation and immunophenotypes of relapsed/refractory primary AML peripheral blood samples were assessed in 96-well plates under four culture conditions: 1) cell culture medium only 2) in direct contact with HS-5 bone marrow stromal cells 3) in HS-5 conditioned medium or 4) in a cocktail of four cytokines as per our previously validated cytokine-based assay. Cell viability, cell count and immunophenotypes by fluorescently-conjugated antibodies against CD33, CD34, CD38, CD45, CD117, and CD90 (stromal marker) were determined by flow cytometry weekly for 2 weeks.Results: Our results demonstrated a very heterogeneous response in the growth of primary AML cells among the different patient samples to support with cytokines, HS-5 conditioned medium, and direct contact with HS-5 stromal cells. Overall, primary AML cells continued to expand after 2 weeks in both cytokine and stromal support. The majority of primary AML samples demonstrated higher proliferation with direct contact with stromal cells compared to cytokines only. AML cells growth with cytokines or HS-5 conditioned medium was also comparable. Our initial investigation also revealed one patient sample with a CD34+/CD38+ predominant phenotype, which demonstrated a > 600-fold expansion within 2 weeks with cytokines only - greater than 3-times the expansion seen by direct contact with HS-5 stromal cells. Immunophenotypes were maintained for all culture conditions at 2 weeks.Conclusions: Unlike previous studies, which often investigated the long-term proliferation of primary AML cells with both cytokines and stromal support, we showed that cytokine support not only maintained, but can also continue to expand primary AML cells after 2 weeks, independent of stromal support. Consistent with previous studies, we also confirmed that HS-5 stromal cells support long-term in-vitro expansion of primary AML cells, which cannot be substituted by HS-5 conditioned medium. As expected, the majority of primary AML cells showed higher proliferation with direct contact with HS-5 stromal cells compared to cytokine support, but based on one of our patient's sample, which showed a significantly higher expansion with cytokine support only, it is possible that there is a subset of patients whose cell growth respond better to cytokines. This study represents the first direct comparison between the effects of cytokines and two different stromal conditions on the long-term growth of primary AML cells in high throughput format, while providing support that these in-vitro culture conditions can be independently utilized to compare the differential primary AML cell response to anti-leukemic agents. DisclosuresFrattini:Lin Bioscience: Consultancy; Astellas: Honoraria.

RGDS-Modified Superporous Poly(2-Hydroxyethyl Methacrylate)-Based Scaffolds as 3D In Vitro Leukemia Model.

Superporous poly(2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) (P(HEMA-AEMA)) hydrogel scaffolds are designed for in vitro 3D culturing of leukemic B cells. Hydrogel porosity, which influences cell functions and growth, is introduced by adding ammonium oxalate needle-like crystals in the polymerization mixture. To improve cell vitality, cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide is immobilized on the N-(γ-maleimidobutyryloxy)succinimide-activated P(HEMA-AEMA) hydrogels via reaction of SH with maleimide groups. This modification is especially suitable for the survival of primary chronic lymphocytic leukemia cells (B-CLLs) in 3D cell culture. No other tested stimuli (interleukin-4, CD40 ligand, or shaking) can further improve B-CLL survival or metabolic activity. Both unmodified and RGDS-modified P(HEMA-AEMA) scaffolds serve as a long-term (70 days) 3D culture platforms for HS-5 and M2-10B4 bone marrow stromal cell lines and MEC-1 and HG-3 B-CLL cell lines, although the adherent cells retain their physiological morphologies, preferably on RGDS-modified hydrogels. Moreover, the porosity of hydrogels allows direct cell lysis, followed by efficient DNA isolation from the 3D-cultured cells. P(HEMA-AEMA)-RGDS thus serves as a suitable 3D in vitro leukemia model that enables molecular and metabolic assays and allows imaging of cell morphology, interactions, and migration by confocal microscopy. Such applications can prospectively assist in testing of drugs to treat this frequently recurring or refractory cancer.

Chronic IL-1 exposure drives LNCaP cells to evolve androgen and AR independence.

Chronic inflammation promotes prostate cancer (PCa) initiation and progression. We previously reported that acute intereluekin-1 (IL-1) exposure represses androgen receptor (AR) accumulation and activity, providing a possible mechanism for IL-1-mediated development of androgen- and AR-independent PCa. Given that acute inflammation is quickly resolved, and chronic inflammation is, instead, co-opted by cancer cells to promote tumorigenicity, we set out to determine if chronic IL-1 exposure leads to similar repression of AR and AR activity observed for acute IL-1 exposure and to determine if chronic IL-1 exposure selects for androgen- and AR-independent PCa cells. We generated isogenic sublines from LNCaP cells chronically exposed to IL-1α or IL-1β. Cells were treated with IL-1α, IL-1β, TNFα or HS-5 bone marrow stromal cells conditioned medium to assess cell viability in the presence of cytotoxic inflammatory cytokines. Cell viability was also assessed following serum starvation, AR siRNA silencing and enzalutamide treatment. Finally, RNA sequencing was performed for the IL-1 sublines. MTT, RT-qPCR and western blot analysis show that the sublines evolved resistance to inflammation-induced cytotoxicity and intracellular signaling and evolved reduced sensitivity to siRNA-mediated loss of AR, serum deprivation and enzalutamide. Differential gene expression reveals that canonical AR signaling is aberrant in the IL-1 sublines, where the cells show constitutive PSA repression and basally high KLK2 and NKX3.1 mRNA levels and bioinformatics analysis predicts that pro-survival and pro-tumorigenic pathways are activated in the sublines. Our data provide evidence that chronic IL-1 exposure promotes PCa cell androgen and AR independence and, thus, supports CRPCa development.

Mechanisms Regulating Myeloma Cell Responsiveness to Antisense Oligonucleotides Targeting IRF4

Functional genomics studies from our group and others, including CRISPR screens, have documented that interferon regulatory factor 4 (IRF4) is a critical transcription factor (TF) for multiple myeloma (MM) cells in preclinical in vitro and in vivo models; and one of the top most pronounced and recurrent dependencies for MM cells vs. other neoplasias. IRF4 lacks known ligand-binding pocket(s) amenable to selective inhibition by small-molecule pharmacological agents. IRF4 is thus considered, as many TFs, as "undruggable". Recent progress in antisense oligonucleotides (ASOs) and their in vivo properties motivated us to systematically evaluate the response of MM cells to anti-IRF4 ASOs, to obtain direct insights into their potential therapeutic applications in MM and also as functional probes into IRF4 biology. We observed that a panel of anti-IRF4 ASOs (but not control ASO) exhibited dose- and time-dependent activity against genotypically diverse MM cell lines (sub-uM IC50s for most MM lines vs. >10uM against non-MM cells lacking IRF4, e.g. HS5 bone marrow stromal cells [BMSCs]). Major in vitro anti-MM activity could be observed within 3-5 days of treatment; and was preceded by significant decrease in intracellular IRF4 protein levels. Importantly, 24-hour exposure to IRF4 ASO (followed by washout and subsequent culture in ASO-free media for up to 6 days) can induce similar anti-MM activity as continuous in vitro exposure over 6 days. Combinations of IRF4-ASO with several established (e.g. proteasome inhibitor, thalidomide derivative, glucocorticoids) or investigational (e.g. venetoclax) agents for MM result led to enhanced or even synergistic effects in a panel of 6 MM cell lines. For some of these MM cell lines, their in vitro response to IRF4 ASO had statistically significant attenuation in co-cultures with BMSCs: this effect was partially recapitulated in MM cell monocultures supplemented by conditioned media from BMSCs or (to a lesser extent) rhIL6 treatment. To obtain insights into cell-autonomous and nonautonomous mechanisms regulating MM cell responses to IRF4 ASOs, we examined the transcriptional profiles of MM.1S cells exposed to ASOs; and also performed genome-scale CRISPR studies to define genes whose editing (loss-of-function, LOF) or activation (gain-of-function, GOF) alter the MM cell response to the IRF4 ASOs. The transcriptional signature of MM cell treatment with IRF4 ASO (vs. control ASO) comprised distinct clusters of downregulated genes with preferential expression in normal or malignant plasma cells vs. other lineages; CRISPR-validated roles as dependencies for MM cells in vitro (preferentially essential for MM vs. non-MM tumors; or pan-essential); and/or proximity to large areas of chromatin accessibility (defined by H3K27Ac genome-wide ChIP-Seq or ATAC-Seq). Importantly, several genes downregulated by IRF4 ASO treatment are upregulated in MM cells cocultured with BMSCs, possibly explaining at least in part the impact of co-cultures on anti-MM activity of ASO. In our integrated genome-scale CRISPR studies (and validation of many genes with individual sgRNAs), MM cell response to IRF4 ASOs was attenuated by LOF of endonuclease RNase H1 (RNASEH1; which degrades the target RNA when it establishes intracellular hybrids with the ASOs); LOF of diverse genes involved in ASO endocytosis (e.g. IGF2R, PICALM, SH3GL1, RAB5C) or regulation of chondroitin/heparan sulfate (SLC35B2, B3GAT3, B4GALT7, ALG5 and ALG6); GOF of IRF4 itself (likely reflecting a "stoichiometric" effect, e.g. induction of higher IRF4 mRNA levels may require higher concentrations of ASO to maintain anti-MM effect); and GOF of exocytosis-related genes(e.g. CLU, QPCT). Importantly, LOF or GOF of individual genes typically associated with high-risk MM was not associated with decreased MM cell response to the IRF4 ASOs: therefore, retaining intracellular accumulation of IRF4 ASO and productive knockdown of IRF4 mRNA remains a primary driver of anti-MM activity of ASOs even in the context of biologically aggressive MM cells with "high-risk" features. Our results provide comprehensive integrated assessment of the molecular and functional landscapes associated with dysregulation of IRF4; have direct implications for our mechanistic understanding of the role of IRF4 in MM biology; and provide a framework for IRF4 targeting by ASOs or potentially other therapeutic approaches. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Luo:Ionis Pharmaceuticals, Inc.: Current Employment. Kim:Ionis Pharmaceuticals, Inc.: Current Employment. MacLeod:Ionis Pharmaceuticals, Inc.: Current Employment. Mitsiades:Arch Oncology: Research Funding; Sanofi: Research Funding; FIMECS: Consultancy, Honoraria; Karyopharm: Research Funding; Abbvie: Research Funding; Takeda: Other: employment of a relative; TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Fate Therapeutics: Consultancy, Honoraria; EMD Serono: Research Funding; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria.

Identification of Novel Combination Therapies Active in BCL2 Inhibitor-Resistant Patient-Derived AML Models

Acute myeloid leukemia (AML) is an aggressive, heterogeneous malignancy. AML patients whose disease relapses on chemotherapy or are unfit for aggressive induction regimens have limited therapeutic options. Many patients benefit from the combination of venetoclax (BCL2i) and a hypomethylating agent (HMA) but this regimen is rarely curative. The addition of novel agents could provide improved benefit for relapsed/refractory patients. To identify such regimens, we screened a panel of 10 AML cell lines with combinations of venetoclax and novel targeted agents. The agents used spanned multiple mechanisms of action (e.g. DNA damage response, kinase signaling, pro-apoptotic agents) and are all in early clinical development. Cells were treated for 72hrs and viability was assessed by CellTiter-Glo. In several of the cell lines that were insensitive or partially sensitive to venetoclax (OCI-AML3, KG1a, MonoMac6, THP1), combinations with inhibitors of MCL1 (AZD5991), AURKB (AZD2811), and BRD4 (AZD5153) showed synergistic activity (Loewe synergy score >5, growth inhibition > 180%) (Table 1). We next asked if these combinations were active in patient-derived xenograft (PDX) models of AML. We established an ex vivo co-culture assay using the HS-5 bone marrow stromal cell line. AML PDX cells were isolated from mouse spleens and plated in 96-well format in direct co-culture with HS-5 cells or in HS-5-derived conditioned media. Cells were treated with three doses of each monotherapy and three doses of fixed ratio combination. Replicate screens using cells from individual mice on different days confirmed data were reproducible (r2=0.687) across animals engrafted with the same PDX. Drug response was similar between conditioned media and direct co-culture assays (r2=0.81). Venetoclax sensitivity varied across PDX models ex vivo. Notably, 2/5 PDX models screened (DFAM-68555 and DFAM-10360) were insensitive to both venetoclax and the combination of venetoclax + 5-azacytidine (HMA) ex vivo. Both models were established from untreated/1L patients and harbor TP53 mutations. Combination treatments did not add additional benefit over venetoclax monotherapy in the DFAM-10360 model. However, in DFAM-68555, AZD5153, AZD5991, and AZD2811 showed improved activity over venetoclax alone (67%, 54%, and 67% vs. 26% decrease in viability for venetoclax alone, respectively). Since combination strategies will likely be most impactful in patients refractory to or relapsed after venetoclax, we chose this venetoclax insensitive model to prioritize in vivo. To confirm the translatability of these findings, we designed a pilot in vivo study using DFAM-68555. Mice were randomized to receive vehicle, venetoclax + HMA, or venetoclax + AZD5153 when peripheral blood disease reached ~5% (hCD45+hCD33+ cells by flow cytometry). After two weeks of dosing, animals were sacrificed to evaluate disease burden in bone marrow (sternum), spleen, and peripheral blood. The model remained insensitive to venetoclax + HMA in vivo. The combination of AZD5153 with venetoclax decreased disease burden in blood and spleen compared to vehicle (30% and 42% hCD45+CD33+ cells by flow cytometry vs 70% and 95%, respectively) with similar efficacy seen by immunohistochemistry in the bone. Finally, we screened these venetoclax combinations in additional aggressive AML PDX models which were resistant or only partially responsive to venetoclax in vivo. Addition of AZD2811NP and AZD5991 to venetoclax was more effective than venetoclax alone and venetoclax + HMA in the bone marrow. The most active combination varied from model to model. Efficacy screening in additional models is ongoing to further build ex vivo to in vivo translation and prioritize development of specific combinations. Also ongoing is genomic and transcriptomic profiling of these PDXs to identify potential predictive biomarkers of combination activity. In summary, we developed an ex vivo screening platform to test clinically actionable combinations for activity in clinically relevant models. Using this platform and subsequent in vivo efficacy, we identified venetoclax combinations across multiple mechanisms (pro-apoptotic, cell cycle regulation, transcriptional regulation, DNA damage response) with activity in venetoclax-insensitive models. These results suggest potential therapeutic options to explore clinically for AML patients. Disclosures Andersen: AstraZeneca: Employment. Christie:AstraZeneca: Employment. Rosen:Astrazeneca: Employment. Maratea:AstraZeneca: Employment. Hattersley:AstraZeneca: Employment. Travers:AstraZeneca: Employment. Cidado:AstraZeneca: Employment. Pulukuri:AstraZeneca: Employment. Saeh:AstraZeneca: Employment. Clark:AstraZeneca: Employment, Equity Ownership. Reimer:AstraZeneca: Employment. Mettetal:AstraZeneca: Employment.

Epigenetic Drug Combination Chemo-Sensitizes Pediatric AML By Reducing Cell Adhesion and Dislodging AML Cells from the Bone Marrow

BackgroundWe showed earlier that prolonged treatment with a combination of azacitidine (DNMT1 inhibitor) and panobinostat (HDAC inhibitor) induced complete remission in a disseminated xenograft model of KMT2A rearranged AML (Gopalakrishnapillai et al., 2017, Leuk Res). Pretreatment with epigenetic drugs has been shown to induce chemo-sensitivity in some malignancies. However, studies on the use of epigenetic drugs in overcoming chemoresistance mediated by the bone marrow microenvironment are limited. The aim of this study was to determine the merit of incorporating epigenetic therapy with chemotherapy in AML treatment regimen and to identify the mechanism by which epigenetic drugs can chemosensitize AML.MethodsAML cell lines and PDX lines were co-cultured with HS5 bone marrow stromal cells. Cell viability following drug treatment was assessed by flow cytometry. For determination of cell adhesion, violet proliferation dye stained AML cells were co-cultured with HS5 cells. Following treatment, non-adherent cells were removed by washes with phosphate buffered saline. The number of adherent AML cells was determined by flow cytometry. NSG-B2m mice were engrafted with MV4;11 cells and bled regularly to evaluate disease progression. Once engraftment was confirmed mice were assigned to treatment groups. Epigenetic therapy constituted azacitidine and panobinostat (2.5 mg/Kg each; Qd5). Chemotherapy constituted cytarabine (50 mg/Kg; Qd5) and daunorubicin (1.5 mg/Kg; Qd3). Mice were euthanized when they reached predetermined experimental endpoints. All animal studies were approved by the Institutional Animal Care and Use Committee.ResultsAML cells in co-culture with HS5 bone marrow stromal cells were less sensitive to chemotherapeutics cytarabine and daunorubicin compared to AML cells in monoculture. This chemoprotection was not achieved when AML cells were cultured in HS5 conditioned media or in Transwell inserts suspended over HS5 monolayers, suggesting that direct cell-to-cell contact was required. MV4;11 cells exposed to epigenetic drugs or cytarabine alone retained 70% viability. Pre-treatment with the epigenetic drug combination of azacitidine and panobinostat prior to cytarabine exposure greatly reduced cell viability in MV4;11 cells harboring KMT2A fusion (Fig. 1A). Similar chemo-sensitization was observed when four distinct KMT2A rearranged PDX lines were used ex vivo. This sensitization was accompanied by reduced binding of AML cells to HS5 cells following treatment with epigenetic drugs (Fig. 1B).We evaluated the efficacy of the epigenetic therapy and chemotherapy combination in disseminated xenograft models of pediatric AML. NSG-B2m mice transplanted with MV4;11 cells via the tail vein were randomly assigned to four groups - 1) vehicle, 2) epigenetic therapy 3) chemotherapy (cytarabine + daunorubicin), and 4) epigenetic therapy followed by chemotherapy. The mice receiving the epigenetic therapy and chemotherapy combination survived significantly longer than mice treated with any other condition (Fig. 1C).To assess the leukemic cell distribution in peripheral blood, bone marrow and spleen following treatment, a cohort of mice receiving epigenetic therapy alone or chemotherapy alone were euthanized a day after treatment concluded. The percentage of leukemic cells in bone marrow and spleen was lower in mice treated with epigenetic therapy than those treated with chemotherapy, consistent with the survival data. Surprisingly, the peripheral blood counts were significantly higher in mice receiving epigenetic drug combination. These results together with our in vitro data indicate that epigenetic therapy induces mobilization of AML cells to the blood stream. This increased availability of AML cells may promote enhanced sensitivity to chemotherapy.ConclusionOur data suggest that direct cell-to-cell contact plays a major role in mediating chemoprotective effects of the bone marrow microenvironment. These chemoprotective effects can be overcome by pretreatment with epigenetic drug combination azacitidine and panobinostat. Epigenetic drugs interfere with AML cell interactions with the microenvironment and dislodge AML cells from their protective niche. Thus, mobilization of AML cells to peripheral blood is a potential mechanism of chemo-sensitization mediated by epigenetic drugs. [Display omitted] DisclosuresKolb:Servier: Membership on an entity's Board of Directors or advisory committees; Roche- Genentech: Membership on an entity's Board of Directors or advisory committees.

Direct Comparison of in-Vitro Growth of Primary Human Acute Myeloid Leukemia (AML) Cells in Stromal and Stromal-Free Conditions

Background: Acute myeloid leukemia (AML) is a very aggressive bone marrow malignancy which carries a poor prognosis despite intensive chemotherapy. The treatment of relapsed and refractory AML remains suboptimal. Although more novel therapies are being introduced for AML, there is a limitation of appropriate, predictive, preclinical models available to identify and test novel therapies. In-vitro drug sensitivity testing of patient-derived AML cells is increasingly being used to facilitate treatment options. However, these tests are often done in suboptimal conditions with difficulty in the interpretation of results. Accumulating evidence has shown that the long-term, in-vitro, survival of primary AML cells can be supported with stromal co-culture, which would also take into account the influence of the surrounding tumor microenvironment. However, there has been no direct comparison of a stromal co-culture method to non-stromal growth method of primary AML cells. This is the first comprehensive direct comparison of the proliferation and immunophenotypes of primary AML cells under two different stromal conditions and cytokines, the results of which would not only highlight the importance of stromal cells to chemotherapy resistance but also lay the groundwork for the feasibility and importance of comparing it to non-stromal drug sensitivity testing.Methods: The proliferation and immunophenotypes of seven bio-banked, relapsed/refractory primary AML peripheral blood samples were assessed in 6-well plates under four culture conditions: 1) cell culture media only 2) in direct contact with HS-5 bone marrow stromal cells 3) in HS-5 conditioned media or 4) in a cocktail of four cytokines. Cell viability, cell count and immunophenotypes by fluorescently-conjugated antibodies against CD33, CD34, CD38, CD45, CD117, and CD90 were determined by flow cytometry weekly for 2 weeks.Results: Our results demonstrated a heterogeneous growth response among the seven patient samples in response to the four growth conditions. However, the end effect was that cytokines, HS-5 conditioned media and HS-5 stromal cells can all support the long-term proliferation and viability of the majority of primary AML samples. These three conditions showed statistically significant higher growth compared to the same primary cells cultured in media alone (p-values < 0.05). The degree of expansion was still significantly higher for those cells with cytokines than those with HS-5 conditioned media or stromal cells. Six of the 7 patient samples expanded with cytokine, up to 4-fold by 2 weeks of culture. In comparison 5 of the 7 patient samples expanded up to 3-fold with HS-5 stromal cells, and 4 of the 7 patient samples also expanded up to 3-fold with HS-5 conditioned media at 2 weeks of culture. For the 4 patients, whose cells were supported by both stromal cells and conditioned media, the degree of expansion was similar. Unexpectedly, one out of 7 patient samples had significantly higher expansion compared to all of the other patients in all growth condition, with a 9-fold expansion in cytokine and conditioned media, and a 12-fold expansion in HS-5 stromal cells. There was also a 3-fold expansion in media alone. Baseline AML immunophenotypes were preserved after 2 weeks of culture for all 4 growth conditions, with minimal differentiation.Conclusions: Unlike previous studies, which often investigated the long-term proliferation of primary AML cells with both cytokines and stromal support, we showed that each experimental condition alone can expand primary AML cells. In addition, cytokine support alone can expand primary AML cells at 2 weeks of culture for the majority of patient samples, independent of stromal support, a finding that has previously not been reported. Consistent with previous studies, our data also supports the conclusion that stromal support maintains long-term in-vitro expansion of primary AML cells. All three experimental conditions showed an overall effect of growth with high viability and maintenance of immunophenotypes at 2 weeks of culture. The direct comparison of drug screening under these three experimental conditions would permit the exploration of the protective effect of the bone marrow microenvironment on AML cells allowing for the identification of individualized therapeutic options, while also allowing for comparison to non-stromal based in-vitro assays. DisclosuresNo relevant conflicts of interest to declare.

Identification of an IL‐1‐induced gene expression pattern in AR+ PCa cells that mimics the molecular phenotype of AR− PCa cells

In immunosurveillance, bone-derived immune cells infiltrate the tumor and secrete inflammatory cytokines to destroy cancer cells. However, cancer cells have evolved mechanisms to usurp inflammatory cytokines to promote tumor progression. In particular, the inflammatory cytokine, interleukin-1 (IL-1), is elevated in prostate cancer (PCa) patient tissue and serum, and promotes PCa bone metastasis. IL-1 also represses androgen receptor (AR) accumulation and activity in PCa cells, yet the cells remain viable and tumorigenic; suggesting that IL-1 may also contribute to AR-targeted therapy resistance. Furthermore, IL-1 and AR protein levels negatively correlate in PCa tumor cells. Taken together, we hypothesize that IL-1 reprograms AR positive (AR+ ) PCa cells into AR negative (AR- ) PCa cells that co-opt IL-1 signaling to ensure AR-independent survival and tumor progression in the inflammatory tumor microenvironment. LNCaP and PC3 PCa cells were treated with IL-1β or HS-5 bone marrow stromal cell (BMSC) conditioned medium and analyzed by RNA sequencing and RT-QPCR. To verify genes identified by RNA sequencing, LNCaP, MDA-PCa-2b, PC3, and DU145 PCa cell lines were treated with the IL-1 family members, IL-1α or IL-1β, or exposed to HS-5 BMSC in the presence or absence of Interleukin-1 Receptor Antagonist (IL-1RA). Treated cells were analyzed by western blot and/or RT-QPCR. Comparative analysis of sequencing data from the AR+ LNCaP PCa cell line versus the AR- PC3 PCa cell line reveals an IL-1-conferred gene suite in LNCaP cells that is constitutive in PC3 cells. Bioinformatics analysis of the IL-1 regulated gene suite revealed that inflammatory and immune response pathways are primarily elicited; likely facilitating PCa cell survival and tumorigenicity in an inflammatory tumor microenvironment. Our data supports that IL-1 reprograms AR+ PCa cells to mimic AR- PCa gene expression patterns that favor AR-targeted treatment resistance and cell survival.

Identification of Fibulin-1 as a Human Bone Marrow Stromal (HS-5) Cell-Derived Factor That Induces Human Prostate Cancer Cell Death.

Previous studies showed that human bone marrow stromal HS-5 cells secreted unidentified factor(s) inducing PCa cell death. Herein, the HS-5-derived factor (HS-5 DF) was characterized and identified. Conditioned media from confluent HS-5 cells were collected and modified for biochemical characteristic testing of HS-5 DF. Cell survival was measured by apoptosis assay and live/dead assay. Fibulin-1 was identified from gel electrophoresis and mass spectrometry. The validation of Fibulin-1 as a HS-5 DF was done by immunoprecipitation (IP) and genetic knockdown by CRISPR/Cas9 system. HS-5 DF was trypsin and heat sensitive, but pH stable. The tentative size of the factor fell between 30 kDa and 100 kDa. TGF-β1 treatment led to a suppression of HS-5 DF activity, a property consistent with bone metastasis in prostate cancer. Examination of TGF-β1 down regulated proteins led to identification of fibulin-1 as a candidate for the DF. IP of Fibulin-1 from HS-5 CM and CRISPR knockdown of Fibulin-1 showed a significant reduction of HS-5 CM-derived PCa cell death. These results strongly support a role for fibulin-1 in HS-5 bone marrow stromal cell induction of PCa cell death. Our data indicate that Fibulin-1 functions as a HS-5 bone marrow stromal cell-derived factor inducing prostate cancer cell death. Prostate 77:729-742, 2017. © 2016 Wiley Periodicals, Inc.

The Bone Marrow Environment Promotes Resistance to Mitoxantrone and Etoposide By Distinct Mechanisms in Pediatric AML

Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. One possible mechanism of chemoresistance is enhanced DNA damage repair. Mitoxantrone and etoposide are standard chemotherapy for AML, both leading to DNA damage by inhibition of topoisomerase II. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main processes for DNA damage repair, with ataxia-telangiectaxia mutated (ATM) kinase and DNA-dependent protein kinase (DNA-PK) as key components, respectively. Both kinases phosphorylate histone H2AX (gH2AX), which facilitates DNA damage repair. Additionally, the bone marrow stromal environment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. To study leukemia-stroma interactions, we used HS5 and HS27A human bone marrow stromal cells. In co-culture studies, we found that stroma-mediated resistance to mitoxantrone was mediated by both stromal soluble factors and cell-cell contact, whereas resistance to etoposide mainly by physical contact with stroma. Further, we recently reported that stromal CYR61 promotes resistance to mitoxantrone, but not etoposide (Long, et al, 2015, Br J Haematol, 170:704).To further study the mechanism underlying stroma-induced chemotherapy resistance, 44 diagnostic AML patient samples from the Children’s Oncology Group were co-cultured on stromal cells, or cultured alone. The samples were treated with 100 nM mitoxantrone (n=27) or 10 µM etoposide (n=32) for 24h. Fifteen samples had sufficient cells for both chemotherapy treatments. Cells were analyzed by FACS, and stromal cells, which express mOrange, and lymphocytes (CD45high, SSClow) were excluded. We measured intracellular levels of cleaved PARP (cPARP) as an apoptosis marker, and gH2AX as a DNA damage signaling marker. As expected, AML cell viability (%cPARP-) after etoposide treatment was significantly higher in the stromal co-cultures (61.2 ±3.3% for AML cells cultured alone, v. 83.2 ±1.8% in HS5 co-cultures, p<0.0001). Results were similar for mitoxantrone treatment. We also found increased DNA damage signaling (%cPARP-/gH2AX+) after mitoxantrone treatment for AML cells in stromal co-culture (34.5 ±3.0% in cells cultured alone, v. 58.6 ±3.1% in HS5 co-cultures, p<0.0001). However, DNA damage signaling was not significantly increased after etoposide treatment. For 5 samples treated with both chemotherapy agents, we also measured pDNA-PKcs and pATM by FACS. Treatment with mitoxantrone, but not etoposide, induced more pDNA-PKcs (MFI, 25.7 ±4.2 untreated, v. 61.7 ±5.1 mitoxantrone, p<0.001, v. 31.5 ±4.8 etoposide) in AML cells cultured alone. However, stroma did not further increase pDNA-PKcs. These results suggest that the NHEJ pathway is important for the repair of DNA damage caused by mitoxantrone, and that stromal cells increase DNA damage signaling by a mechanism not involving increased pDNA-PKcs.To better understand etoposide resistance, 29 of the 44 primary AML samples were cultured on stroma overnight, and activation of intracellular signaling pathways, including pY-STAT3, pY-STAT5, and pERK1/2, was measured by FACS. Responses were heterogeneous overall, but we found several patterns of stroma-induced signaling. For example, a sample that strongly activated a given pathway when co-cultured with HS5 also responded strongly to HS27A cells, and this was particularly true for pY-STAT3 (R=0.71, p<0.0001) and pY-STAT5 (R=0.76, p<0.0001). We also found a correlation between pY-STAT3 and pY-STAT5, such that samples that showed a strong activation of STAT3 with stromal co-culture had a similar activation of STAT5 (R=0.94, p<0.0001, on HS27A). As for signaling pathways in relationship to apoptosis, higher levels of pERK1/2 were associated with lower levels of apoptosis (R=-0.5182, p<0.01, on HS27A), suggesting that ERK1/2 activation may promote resistance to etoposide.In summary, we found that DNA damage signaling is induced in AML cells by mitoxantrone, and it is augmented in cells co-cultured with stroma, likely contributing to mitoxantrone resistance. This mechanism does not occur with etoposide. Instead, we found that stromal environment-induced ERK1/2 signaling may enhance etoposide resistance in pediatric AML patient samples. Further studies to confirm the role of pERK1/2 in stroma-induced etoposide resistance are underway. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4100: Stromalcell suppression of estrogen receptor in growth-arrested breast cancer cellsand promotion of outgrowth of estrogen-independent clones

Abstract Breast cancers have a poorer prognosis if estrogen receptor expression was lost during recurrence. It is unclear whether this conversion is cell autonomous or whether it can be promoted by the microenvironment during cancer dormancy. Bone marrow is the most common site for luminal breast cancer recurrence. We explored whether the HS5 bone marrow stromal cell line that is known to arrest co-cultured breast cancer cells could suppress estrogen receptor alpha (ER) expression during arrest, and whether it could prime the emergence of estrogen-independent breast cancer clones. We demonstrated that paracrine signaling from HS5 cells downregulated ER at the mRNA level and also decreased ER protein stability in T47D and MCF7 breast cancer cells. Conditioned medium (CM) from HS5 arrested the cells in G0/G1 in part through interleukin-1 (IL1), inhibiting mammosphere formation and 2D-growth despite activation of proliferative pathways (Erk and AKT) by the CM. Reversal of stromal-induced growth arrest by blockade of signaling by IL1 in HS5-CM enabled the outgrowth of ER-negative breast cancer cells that were fulvestrant-resistant and estrogen-independent. Outgrowth depended on AKT signaling. Our results describe pro- and anti-proliferative paracrine signals arising from a bone marrow stromal cell line. Blockade of the growth suppressive signaling unmasked stromal facilitation of ER conversion and anti-estrogen resistance in breast cancer cells. Citation Format: Richard Steinman, Jingjing Huang, Paul Woods, Daniel Normolle, Julie Goff, Christine Stehle. Stromalcell suppression of estrogen receptor in growth-arrested breast cancer cellsand promotion of outgrowth of estrogen-independent clones. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4100.

Microenvironmental-Mediated Regulation of L-Selectin in Chronic Lymphocytic Leukaemia

Abstract Chronic Lymphocytic Leukaemia (CLL) is characterized by the accumulation of CD5/19+ve B cells in the blood, bone marrow and lymph nodes. The tumour microenvironment of the bone marrow and secondary lymphoid organs promotes cell proliferation, survival and protection from drug induced apoptosis. Selectins, integrins and chemokines mediate the trafficking of CLL cells to these protective niches. An important adhesion molecule in this process is L-selectin (CD62L) which is involved in initial tethering and rolling of CLL cells in the high endothelial venules, allowing migration between the blood and the secondary lymphoid organs. It has been shown that CLL cell expression of CD62L increases during in vitro culture and blocking antibodies cause an increase in apoptosis, suggesting a role for CD62L in cell survival (Burgess et al., Clin Cancer Res. 2013). The B-cell receptor (BCR) signalling pathway is the most important pathway involved in micro-environmental crosstalk and CLL cell survival, and has recently been shown to interact with the STAT3 signalling pathway (Rozovski et al., Blood 2014). In this study, Hs5 Bone Marrow Stromal Cell (BMSC), Human Umbilical Vein Endothelial cells (HUVEC) and patient derived BMSC co-cultures were used to mimic pro-survival microenvironmental signals and investigate the effect on the expression of a panel of cell surface adhesion molecules. These coculture models resulted in a significant increase in CD62L positive CLL cells (p&lt;0.01, n=7). Stimulation of the BCR using immunoglobulin F(ab´)2fragments induced tyrosine phosphorylation of STAT3 and resulted in a significant increase in CD62L expression, which was abrogated by pre-treatment with the Bruton tyrosine kinase (btk) inhibitor Ibrutinib prior to BCR stimulation. (p&lt;0.05, n=5). siRNA mediated STAT3 knockdown and treatment with STAT3 inhibitors, including Cucurbitacin I, resulted in a significant decrease in CD62L positive CLL cells (p&lt;0.0001, n=14). Co-culture with Hs5 cells, HUVECs, patient derived BMSC or BCR stimulation did not overcome Cucurbitacin I induced downregulation of CD62L. The effect of STAT3 inhibition on CLL cell adhesion and chemotaxis was also investigated. A microfluidic system including a neMESYS Low Pressure syringe pump system (Cetoni GmbH) and Cellix Vena8Endothelial+biochips coated with Human Umbilical Vein Endothelial cells (HUVECs) or Human Dermal Lymphatic Endothelial cells (HDLEC) was utilised to investigate CLL cell adhesion under fluid shear flow conditions. Flow rates of0.5 dynes/cm2 for the HUVEC-coated biochips and 0.08 dynes/cm2 for the HDLEC coated biochips were used to model the fluid shear of blood and lymphatic flow respectively. Treatment with Cucurbitacin I resulted in a mean decrease of 50% in CLL cell adhesion to HUVEC but not HDLEC in this system (p&lt;0.05, n=3). Chemotaxis of CLL cells was investigated using Neuroprobe 96-well ChemoTx plates. Treatment with Cucurbitacin I resulted in a mean decrease of 87% of CLL cells migrated in response to the chemokine CXCL12 compared to control (p&lt;0.0001, n=4). In conclusion, this study shows microenvironmental signals mediate the expression of CD62L in CLL cells. Our data suggest that the STAT3 pathway regulates the expression of CD62L and the adhesion and chemotaxis of CLL cells. The down-regulation of CD62L expression through inhibition of BCR signalling or direct inhibition of STAT3 may have implications for the trafficking of CLL cells and treatment of CLL. Disclosures No relevant conflicts of interest to declare.

Transforming growth factor-β signaling induced during prostate cancer cell death and neuroendocrine differentiation is mediated by bone marrow stromal cells.

Prostate cancer that has metastasized to bone undergoes critical interactions with bone marrow stromal cells (BMSCs), ultimately promoting tumor survival. Previous studies have shown that BMSCs secrete factors that promote prostate cancer apoptosis or neuroendocrine differentiation. Because of the significance of transforming growth factor-β (TGF-β) family cytokines in cytostasis and bone metastasis, the role of TGF-β signaling in the context of prostate cancer-BMSC interactions was investigated. The role of TGF-β family signaling in BMSC-induced apoptosis of lineage-related prostate cancer cells was investigated in live/dead assays. SMAD phosphorylation or activity during apoptosis and neuroendocrine differentiation was investigated using immunofluorescence, Western blotting, and luciferase reporter assays, along with the ALK-4, -5, -7 kinase inhibitor, SB-431542. Treatment of castration-resistant prostate cancer cells with SB-431542 resulted in significant reduction of apoptosis mediated by HS-5 BMSCs, supporting the involvement of TGF-β/SMAD signaling during this event. Interestingly, however, pre-treatment of BMSCs with TGF-β1 (5 ng/mL) yielded a conditioned medium that elicited a marked reduction in prostate cancer death. Phosphorylated-SMAD2 (P-SMAD2) was activated in BMSC-triggered transdifferentiated prostate cancer cells, as demonstrated through immunoblotting and luciferase reporter assays. However, SB-431542 did not restore androgen receptor and prostate specific antigen levels down-regulated by BMSC-secreted factors. Prostate cancer cells induced to undergo neuroendocrine differentiation in a BMSC-independent mechanism also showed elevated levels of P-SMAD2. Collectively, our findings indicate that: (1) TGF-β family cytokines or regulated factors secreted from BMSCs are involved in prostate cancer apoptosis; (2) TGF-β signaling in prostate cancer cells is induced during neuroendocrine differentiation; and (3) TGF-β1 stimulation of BMSCs alters paracrine signaling to create a permissive environment for prostate cancer survival, suggesting a mechanism for prostate cancer-mediated colonization of bone. TGF-β signaling resulting in activation of SMAD2 in prostate cancer may be an indicator of cellular stress in the presence of toxic paracrine factors released from the bone marrow stroma, ultimately fostering prostate cancer colonization of bone.

Abstract 4031: CDK4/6 inhibition amplifies IMiD killing of myeloma cells by accelerating CRL4CRBN-dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2

Abstract The immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) are effective therapies for hematologic malignancies, especially multiple myeloma (MM). Cereblon (CRBN) is required for IMiD's anti-myeloma activity. Emerging evidence has established that CRBN is a substrate receptor of the CUL4-ROC1-DDB1-CRBN (CRL4CRBN) E3 ligase complex and that the homeobox transcription factor MEIS2 is an endogenous substrate of CRL4CRBN. IMiDs block MEIS2 from binding to CRBN, thereby facilitating the recruitment of transcription factors IKZF1 and IKZF3 to CRL4CRBN for ubiquination-proteasome degradation. IKZF1 and IKZF3 appear to be required for MM survival, in part by transcriptional regulation of IRF4. These findings, obtained in heterologous systems and MM cell lines, suggest that modulation of the CRL4CRBN E3 ligase is a critical early event in IMiD killing of MM cells. We investigated its physiologic relevance in primary bone marrow myeloma cells (BMMCs) in co-culture with HS-5 bone marrow stromal cells. We showed that IMiDs preferentially kill BMMC in G1 arrest and that induction of prolonged early G1 arrest (pG1) by selective inhibition of CDK4/CDK6 with palbociclib (PD 0332991) markedly enhanced Len/Pom killing of BMMCs (14/21), unless the BMMCs were refractory or extremely sensitive to Len (n = 7). Importantly, the IMiD sensitivity in BMMCs ex vivo correlates with subsequent clinical responses to IMiD-based therapies in vivo in 7/7 cases characterized. This novel finding suggests that although IMiDs target multiple cell types, the intrinsic IMiD sensitivity is a major determinant for the clinical response to IMiDs in MM. Whole transcriptome sequencing and gene set enrichment analysis further revealed that Len induces the interferon response signature genes in BMMCs, and this is enhanced by palbocicib. While CRBN is ubiquitously expressed, IKZF3 and IRF4 (mRNA and protein) and the IKZF1 protein were barely detectable in normal bone marrow plasma cells and prominently increased in BMMCs. Primary myeloma cells thus appear to be addicted to IKZF1/IKZF3-IRF4 signaling for survival, and IMIDs induce CRL4CRBN-mediated degradation of IKZF1 and IKZF3 to antagonize this survival pathway. Accordingly, palbociclib enhancement of Len killing was associated with rapid acceleration of IKZF1/IKZF3 degradation, within one hour of Len treatment in BMMCs. Palbociclib acts in opposition of MEIS2, because MEIS2 knockdown mimicked palbociclib sensitization and MEIS2 reconstitution antagonized palbociclib sensitization. In summary, we have demonstrated that induction of pG1 by CDK4/6 inhibition sensitizes primary myeloma cells to IMiD killing through rapid acceleration of CRL4CRBN -dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2. These findings are being validated in a clinical trial targeting CDK4/6 with palbociclib in combination with Len in MM. Note: This abstract was not presented at the meeting. Citation Format: Xiangao Huang, David Jayabalan, Maurizio Di Liberto, Tomer M. Mark, Zhengming Chen, Adriana Rossi, Anna C. Schinzel, William C. Hahn, Mohamad A. Hussein, Rubin Niesvizky, Selina Chen-Kiang. CDK4/6 inhibition amplifies IMiD killing of myeloma cells by accelerating CRL4CRBN-dependent degradation of IKZF1 and IKZF3 in opposition to MEIS2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4031. doi:10.1158/1538-7445.AM2015-4031

Abstract B15: Bone marrow stromal cell-secreted inflammatory cytokines promote treatment resistance and survival of prostate cancer cells

Abstract It is well established that prostate cancer (PCa) cells hone to, adapt, and thrive in the bone. Importantly, bone metastases are found in over 80% of PCa deaths. Therefore, it is imperative to elucidate and characterize the molecular mechanisms that enable PCa cells to thrive in the bone and resist conventional PCa therapies, such as hormone ablation or anti-androgens. We found that the HS-5 bone marrow stromal cell (BMSC) line, a secretory BMSC line that can support hematopoietic stem cell growth, secretes paracrine factors that induce apoptosis in PCa cells. However a subpopulation of the PCa cells can survive the BMSC-induced death. The surviving PCa subpopulation differentiates into a neuronal morphology and looses androgen receptor (AR) expression, reminiscent of treatment-resistant neuroendocrine PCa. Ideally, the loss of AR would induce PCa cell death; however we observe a concomitant increase in the autophagy-related cell survival protein, p62/SQSTM1, and induction of cytoprotective autophagy. Furthermore, we find that interleukin-1 beta (IL-1β) and IL-6, inflammatory cytokine secreted by HS-5 BMSCs, are sufficient to promote PCa neuronal morphology, reduce AR accumulation, upregulate p62/SQSTM1 levels, and induce autophagy. In addition, genetic silencing of p62/SQSTM1 causes PCa cell death and pharmacological inhibition of cytokine or autophagy signaling attenuates PCa neuroendocrine differentiation. Thus, our PCa-bone marrow stromal cell model suggests that PCa cells can co-opt the immune system in the bone microenvironment leading to PCa androgen independence, treatment resistance, and survival in the bone. Furthermore, our data suggests that inhibiting inflammatory cytokine signaling and/or the autophagy process are rational strategies to combat PCa bone metastasis. Citation Format: Megan Chang, Micaela Morgado, Viral Patel, Michael Gwede, Mary Cindy Farach-Carson, Nikki Delk. Bone marrow stromal cell-secreted inflammatory cytokines promote treatment resistance and survival of prostate cancer cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B15. doi:10.1158/1538-7445.CHTME14-B15

Loss of the Histone Demethylase UTX Contributes to Multiple Myeloma and Sensitizes Cells to EZH2 Inhibitors

Genetic alterations of epigenetic regulators have become a recurrent theme in hematological malignancies. In particular, aberrations that alter the levels or distribution of methylation of lysine 27 on histone H3 (H3K27me) have emerged as a common feature of a wide variety of cancers, including multiple myeloma (MM). The histone demethylase UTX/KDM6A activates gene expression by removing the H3K27me3 repressive histone mark, counteracting the activity of EZH2, the enzyme that places this modification. UTX somatic inactivating mutations and deletions are found in up to 10% of MM cases; nevertheless, the epigenetic impact of UTX loss in MM and the mechanisms by which it contributes to this disease remain to be elucidated.To ascertain the biological impact of UTX loss, we used a recently identified isogenic cell line pair: ARP-1 (UTX wild-type) and ARD (UTX null). UTX-null ARD cells were engineered to express UTX in a doxycycline-inducible manner. UTX add-back slowed the proliferation rate of ARD cells, without affecting their viability. Soft agar assays demonstrated that UTX-null ARD cells have increased clonogenicity compared to UTX-wild-type ARP-1 cells. Re-expression of UTX partially reversed this effect, decreasing the number and size of colonies formed. ARD cells also showed increased adhesion to Hs-5 bone marrow stromal cells and to fibronectin than ARP-1 cells, an ability associated with cell survival and drug resistance. UTX add-back decreased the adhesive properties of ARD cells demonstrating this effect is dependent on UTX loss.Mass spectrometry analysis of the add-back system and a panel of UTX wild-type and mutant MM cell lines showed that global levels of H3K27me are not altered after UTX loss or upon its add-back. Therefore, UTX depletion may alter H3K27me at specific loci, and control the expression of a limited number of genes. To identify the genes and pathways that are altered upon UTX loss, we performed RNA-sequencing (RNA-seq) on the paired MM cell lines and the add-back system. This analysis revealed approximately 5,000 genes differentially expressed between ARP-1 and ARD cells. Re-expression of UTX in the UTX-null ARD cells reversed the expression of approximately 1,400 genes, most of them being upregulated upon reintroduction of UTX. Gene ontology analysis of genes responsive to UTX manipulation identified pathways such as JAK-STAT, cadherin, integrin and Wnt pathways. Many of these pathways are related to cell adhesion properties, correlating with the effects observed in vitro. Some examples of the genes which expression was restored upon UTX add-back are E-cadherin, whose loss has been associated with MM progression; and PTPN6, a negative regulator of the JAK-STAT pathway. Chromatin immunoprecipitation (ChIP) experiments at UTX target genes revealed a decrease in H3K27me3 and a concomitant increase in H3K4me3 upon UTX add-back, correlating with the observed changes in gene expression.As loss of UTX leads to a failure in the removal of H3K27me3, we hypothesized that UTX-null cells may be more dependent on EZH2 to maintain high H3K27me3 levels at specific loci. Treatment of the paired cell lines with the EZH2 inhibitor GSK343 for 7 days significantly decreased the viability of UTX-null ARD cells, but had no effect on the UTX wild-type ARP-1 cells. This effect was not exclusive to these cell lines, as treatment of a panel of UTX wild-type and mutant MM cells corroborated the increased sensitivity in UTX-mutant cells. RNA-seq of ARD cells treated with GSK343 for 7 days identified approximately 2,000 genes with altered expression in response to this drug, most of them being upregulated upon EZH2 inhibition. These genes partially overlapped with the genes that were responsive to UTX add-back, including E-cadherin, suggesting that treatment with EZH2 inhibitors is somewhat similar to UTX add-back.Collectively, this work demonstrates that loss of UTX alters the epigenetic landscape of MM cells, leading to altered expression of a specific set of genes, ultimately benefiting cells through increased proliferation, clonogenicity and adhesion. Moreover, inhibition of EZH2 partially reverses aberrations promoted by UTX loss and may represent a rationale therapy for the treatment of this type of MM. DisclosuresNo relevant conflicts of interest to declare.

IL-1β induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells.

Chronic inflammation is associated with advanced prostate cancer (PCa), although the mechanisms governing inflammation-mediated PCa progression are not fully understood. PCa progresses to an androgen independent phenotype that is incurable. We previously showed that androgen independent, androgen receptor negative (AR(-) ) PCa cell lines have high p62/SQSTM1 levels required for cell survival. We also showed that factors in the HS-5 bone marrow stromal cell (BMSC) conditioned medium can upregulate p62 in AR(+) PCa cell lines, leading us to investigate AR expression under those growth conditions. In this paper, mRNA, protein, and subcellular analyses reveal that HS-5 BMSC conditioned medium represses AR mRNA, protein, and nuclear accumulation in the C4-2 PCa cell line. Using published gene expression data, we identify the inflammatory cytokine, IL-1β, as a candidate BMSC paracrine factor to regulate AR expression and find that IL-1β is sufficient to both repress AR and upregulate p62 in multiple PCa cell lines. Immunostaining demonstrates that, while the C4-2 population shows a primarily homogeneous response to factors in HS-5 BMSC conditioned medium, IL-1β elicits a strikingly heterogeneous response; suggesting that there are other regulatory factors in the conditioned medium. Finally, while we observe concomitant AR loss and p62 upregulation in IL-1β-treated C4-2 cells, silencing of AR or p62 suggests that IL-1β regulates their protein accumulation through independent pathways. Taken together, these in vitro results suggest that IL-1β can drive PCa progression in an inflammatory microenvironment through AR repression and p62 induction to promote the development and survival of androgen independent PCa.

Down-Regulation of CD9 by Methylation Decreased Bortezomib Sensitivity in Multiple Myeloma

Bortezomib therapy has been proven successful for the treatment of relapsed and/or refractory multiple myeloma (MM). However, both intrinsic and acquired resistance has already been observed. In this study, we explored the relationship between CD9 expression and bortezomib sensitivity in MM. We found that down-regulation of CD9 by methylation decreased bortezomib sensitivity in multiple myeloma. CD9 expression obviously increased bortezomib sensitivity through inducing apoptosis, significantly inhibiting U266 cells' adhesion to HS-5 and primary bone marrow stromal cells, but increasing U266 cells' adhesion to fibronectin. CD9 expression also significantly inhibited U266 cell migration. The mechanisms may include: the endoplasmic reticulum stress pathway, cell adhesion related signaling pathway and osteoclast differentiation related signaling pathway. Combination therapy with de-methylation reagent 5-Aza-2-deoxycytidine may prove useful to the development of novel strategies for the treatment of bortezomib-resistant MM patients.

Pre-Clinical Analysis Of The Novel Antifolate Pralatrexate In Multiple Myeloma Reveals Functional Biomarkers Correlated With Response

Multiple myeloma (MM) is a neoplasia of plasma cells and is the second most common hematologic malignancy. Current combination therapies for MM typically include proteasome inhibitors (PIs) and/or immunomodulatory drugs (ImiDs). Although these regimens are effective, relapse and resistance to these agents are inevitable. There is a need to develop new classes of drugs with activity against MM and to identify biomarkers associated with response to these agents, in order to tailor salvage therapy for individuals most likely to respond and minimize exposure to therapies that are unlikely to be of benefit. We investigated these principles with the novel antifolate pralatrexate (PDX) in laboratory models of MM. Antifolates such as methotrexate (MTX) are not commonly used in the treatment of MM; however, combination chemotherapy regimens containing MTX have demonstrated clinical efficacy. In addition, case reports of the application of intrathecal MTX have shown activity against MM in the central nervous system. PDX was designed to be a preferred substrate for the reduced folate carrier (RFC) and folylpolyglutamate synthetase (FPGS) as compared to MTX, and it is approved for the treatment of peripheral T cell lymphoma. PDX induced intrinsic apoptosis with greater potency than MTX in a subset of human myeloma cell lines (HMCLs). Interestingly, other HMCLs were resistance to antifolate-induced apoptosis. This dichotomy among HMCLs provided a platform to identify functional biomarkers associated with sensitivity or resistance to PDX-induced apoptosis. We examined expression of the genes that are determinants of antifolate actions: RFC, FPGS, gamma-glutamyl hydrolase (GGH), and dihydrofolate reductase (DHFR) in sensitive and resistant HMCLs. Higher pre-treatment levels of RFC mRNA by qRT-PCR correlated with increased sensitivity to PDX and higher rates of RFC-mediated transport. We then quantified mRNA for folate metabolism genes by qRT-PCR in total RNA from CD138-selected MM cells from patients and detected a broad range of expression for RFC, indicating that further studies with primary MM cells are warranted to assess RFC expression as a biomarker of antifolate sensitivity. Interestingly, PDX-resistant HMCLs also demonstrated dose-dependent increases in DHFR protein, one of the major antifolate targets, after treatment whereas PDX-sensitive HMCLs did not. Messenger RNA expression was unchanged under these conditions, suggesting a translational or post-translational mechanism for this activity. Sensitivity to PDX-induced apoptosis was not suppressed by co-incubation with recombinant human interleukin-6 or by co-culture with HS-5 bone marrow stromal cells. Finally, we used a previously validated bioluminescent xenograft mouse model to examine the effects of PDX in vivo. PDX-sensitive MM.1s cells stably-transduced with a dual cassette construct expressing both green fluorescent protein (GFP) and luciferase were introduced by tail-vein injection into NOD/Shi-scid/IL-2Rγnull (NOG) mice. These cells engrafted in the marrow of the femur, pelvis, vertebral bodies, and sternum, recapitulating human disease, and tumor burden was quantified by in vivo imaging after luciferin injection. PDX treated mice demonstrated reductions in tumor burden compared to vehicle treated controls. These results demonstrate that the novel antifolate PDX has potent activity against a subset of HMCLs in vitro and in vivo. Baseline mRNA expression of RFC and post-treatment protein levels of DHFR correlated with response in HMCLs. These findings support further clinical studies to assess the potential role of PDX in the treatment of MM coupled with correlative studies to validate the clinical utility of the biomarkers identified in HMCLs. PDX may be an effective addition to individualized therapy for relapsed multiple myeloma. Disclosures: O'Connor: Spectrum Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Allos Pharmaceuticals: Consultancy.

GLI2 Transcription Factor Modulates CD40 Ligand Expression In Bone Marrow Stromal Cells

The interaction between tumor cells and their surrounding microenvironment is essential for the growth and persistence of cancer cells within the host. Cytokines play a key role in mediating this crosstalk between malignant cells and the tumor microenvironment. Although this interaction is clearly established, many of the molecular mechanisms mediating these signaling events remain elusive. In previous work, we have shown that the cytokine CCL5 can regulate the expression and activity of the transcription factor GLI2 in stromal cells. GLI2 is an effector of hedgehog (HH) signaling; however, our studies reveal a role for GLI2 independent of HH. GLI2, in turn, increases the expression and secretion of IL-6 in the tumor microenvironment, highlighting a novel role for GLI2 in directly modulating cytokine genes. We therefore screened for additional cytokines regulated by this axis in HS-5 bone marrow stromal cells using expression arrays. We found that GLI2 can modulate the expression of several cytokines including CD40 ligand (CD40L), CCL2, CCL7 and CXCL11. CD40L is an immunomodulatory and proinflammatory molecule that has been shown to play a role in multiple hematologic malignancies including chronic lymphocytic leukemia (CLL), Waldenström macroglobulinemia (WM) and acute myeloid leukemia (AML) pathogenesis. We first characterized the regulation of CD40L, because of the importance of CD40L in malignant cell biology. Gene expression results were validated by RT-PCR in two additional bone marrow stromal cell lines Saka and L88 where overexpression of GLI2 resulted in increased CD40L expression. Furthermore, we detected increased levels of soluble CD40L (sCD40L) in L88 and Saka cells overexpressing GLI2. Conversely, knockdown of GLI2 resulted in reduced expression and secretion of this cytokine. Biochemical mapping of the domains critical for this GLI2 regulatory mechanism reveals that N-terminal transcriptional domain is dispensable, as a mutant form of GLI2 lacking this domain (delta N-GLI2) increases CD40L gene expression by RT-PCR, protein expression by western blot and sCD40L secretion by ELISA, beyond that of the full-length GLI2 levels. Bioinformatics analysis of the CD40L promoter indicates the presence of 3 candidate binding sites for GLI proteins, suggesting the modulation of CD40L by GLI2 is mediated by direct interaction with its promoter. Ongoing experiments are aimed at determining the mechanism by which GLI2 regulates CD40L expression (either by direct binding to the CD40L promoter or through another indirect pathway). Together, our data identify a novel mechanism controlling CD40L in stromal cells in the bone marrow microenvironment. Because of the importance of CD40L in malignant cell biology, understanding the mechanism of its regulation is of great importance for the development of therapies aimed at targeting the malignant bone marrow tumor microenvironment. Disclosures:No relevant conflicts of interest to declare.