ALBERT

Description: Abstract 2468 In multiple myeloma (MM) and other hematologic malignancies, bone marrow stromal cells (BMSCs) confer resistance to diverse conventional or investigational therapeutics. During the last decade, data from many groups have concurred that the in vitro anti-MM activity of the proteasome inhibitor bortezomib is very similar in the presence and absence of BMSCs, including primary and immortalized BMSCs. These well-validated observations have supported the notion that novel, more effective, therapies for the treatment of MM should ideally be, similarly to bortezomib, capable of overcoming the protective effect of BMSCs. Interestingly, however, we have observed that primary CD138+ MM tumor cells isolated from patients with clinical refractoriness to bortezomib occasionally exhibit substantial in vitro response to clinically achievable concentrations of this drug. We therefore hypothesized that, under certain previously under-explored experimental settings, BMSCs may alter the threshold of MM cell response to bortezomib-induced apoptosis. To address this hypothesis in conditions that better simulate the clinical context, we conducted compartment-specific bioluminescence imaging (CS-BLI) assays to evaluate the effect of bortezomib on tumor cells co-cultured with BMSCs for different time periods prior to bortezomib administration. We observed that prolonged tumor-stromal co-culture (48–96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for several MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Prolonged co-culture of OPM1, RPMI-8226-Dox40, OCI-My5, KMS12BM and KMS18 cells prior to bortezomib treatment enhanced its activity. Importantly, extended co-culture of MM cell lines MM.1S and MM.1R with BMSCs prior to drug treatment induced significant attenuation of their response to bortezomib, as evidenced by 2–3 fold increase of IC50 values in several independent replicate experiments and a mean % area under the bortezomib dose response curve (AUC) of 5.82% vs 14.10% in the absence vs. presence of BMSCs, respectively (p=0.0079). Consistent with these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells mixed with Luc- BMSCs did not show statistically significant reduction in MM burden with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.1320), as quantified by bioluminescence imaging. In contrast, the same dose and schedule of bortezomib treatment significantly suppressed tumor burden, compared to vehicle-treated controls, of monotypic s.c. xenografts of Luc+ MM.1S cells in SCID mice (p=0.0022), as in prior experience. To evaluate the molecular mechanisms of cell non-autonomous decrease in MM cell response to bortezomib, we compared the transcriptional profiles of MM.1S cells in extended co-cultures with HS-5 BMSCs vs. MM.1S cells cultured in isolation. These studies identified a distinct transcriptional signature of stroma-induced transcripts, including several (e.g. PSMC3, ITGB7, FOS, ALDH1L2) for which transcript expression higher than the median levels for refractory MM patients correlated with shorter overall survival (p

Description: Abstract 3995 Introduction: Multiple myeloma (MM) cells suppress osteoblast (OB) maturation and function and induce osteoclast-mediated bone resorption. However, immature cells of the osteoblast lineage (e.g. pre-osteoblasts) remain present in the MM bone microenvironment and their impact on MM cell response to treatment has not been fully characterized. We therefore studied human immortalized cells of the osteoblast lineage as surrogate models for in vitro studies on the effects of pre-osteoblasts on MM cell proliferation, survival and drug resistance. Methods: Immortalized hFOB 1.19 (here referred to as hFOB) and HOBIT cells were utilized in our studies, because, in the absence of Dexamethasone or vitamin D, they express markers consistent with pre-osteoblast stage of differentiation (high type I collagen levels; and low alkaline phosphatase, osteocalcin and osteopontin expression). Monolayer cultures of hFOB cells were performed to determine drug concentrations that did not exhibit cytotoxic effects on these pre-osteoblasts. Next, co-cultures of hFOB and HOBIT cells were performed using a panel of tumor cell lines from MM (n=13), breast (n=1) and lung (n=1) cancer. Compartment-specific bioluminescence imaging (CS-BLI) assays were undertaken to quantify the proliferative responses of the tumor cells in the presence of pre-osteoblasts, and if any changes are observed in tumor cell responses to established or novel therapeutic agents. Assays with Transwell insert chambers were conducted to allow for culture of tumor cells proximate to pre-osteoblasts, but preclude their direct cell-to-cell contact, in order to determine whether paracrine factors released by pre-osteoblasts are sufficient to recapitulate the effect(s) of their co-culture with tumor cells. Finally, cytokine or cytokine receptor neutralization studies with monoclonal antibodies or selective kinase inhibitors were performed to investigate the functional contribution of IL-6 or IGF1R-mediated signalling in tumor-pre-osteoblast interactions. Results: Pharmacologically relevant doses of conventional and novel therapies did not exhibit substantial cytotoxic effects on hFOB cells. Within 48hrs of co-culture, pre-osteoblasts stimulated proliferation of several tumor cell lines, including MM.1S, MM.1R, RPMI8226 and Dox40 in co-culture with hFOB; and NCI-H929, OPM2, H23 and Dox40 with HOBIT co-culture. hFOB cells decreased the sensitivity of 8 MM cell lines to at least one established (doxorubicin, dexamethasone, lenalidomide) or novel (vorinostat, pomalidomide) therapy tested. These observations were particularly pronounced in MM.1S and KMS34 cell lines. Subsequent Transwell-based co-culture assays showed that lack of direct cell-to-cell contact significantly decreases or even completely abrogates the resistance observed in respect to doxorubicin, lenalidomide or vorinostat in mono-cultures of tumor cells with pre-osteoblasts. Finally, IL-6 neutralization with monoclonal antibody did not overcome the proliferative effect of hFOB on MM cells. The anti-MM effect of IGF1R kinase inhibitors was also suppressed by co-culture with hFOB cells. These 2 latter results taken together indicate that the protection provided by hFOB cells is independent of IL-6 and IGF-1R-signaling in tumor or pre-osteoblasts. Conclusion: Our results suggest that cells of the osteoblast lineage may play a role as promoters of MM cell survival and resistance to diverse established and investigational therapeutics. Ongoing mechanistic and functional validation studies aim to delineate new therapeutic strategies to overcome pre-osteoblast-mediated drug resistance in MM and, potentially, other neoplasias. Disclosures: Richardson: Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees; Oncopep: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor Other.

Description: 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. Disclosures McMillin: 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.

Description: Abstract 1409 MYC is among the most prolific oncogenes in cancer, yet pharmacologic strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have undertaken to target c-Myc transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain-containing proteins as transcriptional regulatory factors for c-Myc. BET inhibition with JQ1 rapidly downregulates c-Myc transcription, followed by depletion of chromatin-bound c-Myc and genome-wide downregulation of Myc-dependent target genes. In translational model systems of multiple myeloma and Burkitt's lymphoma, both canonical MYC -dependent hematologic malignancies, JQ1 treatment produces a potent antiproliferative effect associated with cell cycle arrest. In multiple myeloma, adhesion to bone marrow stroma is associated with upregulation of BRD4, a BET bromomdomain coactivator protein. Inhibition of Myc function with JQ1 leads to impaired adhesion to stroma and cellular senescence, a classical Myc-specific phenotype. Mechanistically, JQ1 treatment depletes BRD4 from IgH enhancers, leading to prompt and robust downregulation of MYC transcription. In vivo efficacy of JQ1 in two disseminated models of multiple myeloma and in a Burkitt's lymphoma human xenograft establishes the therapeutic rationale for BET bromodomain inhibition in these diseases. Together, these studies identify a mechanistic rationale for targeting c-Myc in human cancer, and potentially other undruggable oncogenes driven by immunoglobulin rearrangement. Note: G.C.I. and J.E.D. have made equal contributions to this research; C.S.M. and J.E.B. are jointly senior authors Disclosures: Richardson: Millennium: Advisory Board; Celgene: Advisory Board; Johnson & Johnson: Advisory Board; Novartis: Advisory Board; Bristol Myers Squibb: Advisory Board. Ghobrial:Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celgene:; Novartis:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar:; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding. Bradner:Acetylon Pharmaceuticals: Scientific Founder; SHAPE Pharmaceuticals: Scientific Founder; Tensha Therapeutics: Scientific Founder.

Description: Abstract 2923 Midostaurin (PKC412; Novartis Pharmaceuticals) is a multi-targeted kinase inhibitor currently being evaluated in clinical trials in acute myelogenous leukemia (AML), because of its potent activity in cells expressing mutant FLT3. Prior preclinical studies from our groups have shown that PKC412 has FLT3-independent anti-MM activity, and the effects on AML cells is suppressed by the presence of conditioned media from bone marrow stromal cells (BMSCs), such as the immortalized BMSC line HS-5 (Weisberg et al. Mol Cancer Ther 2007). In this study, we evaluated whether the microenvironment-dependent drug resistance to PKC412 applies to not only AML cells, but also to cells from MM and other FLT3-negative malignant cells. We tested a panel of cells from MM (n=8), FLT-ITDneg AML (n=1), CML (n=2) and breast cancer (n=2) for their response to PKC412 in the presence or absence of BMSCs and other non-malignant accessory cells using tumor cell compartment-specific bioluminescence imaging (CS-BLI), as in our antecedent studies (McMillin et al. Nat Medicine 2010). We also compared the PKC412 response of the aforementioned neoplastic cells when cultured in vitro in the presence or absence of conditioned media (CM) from different types of BMSCs known to confer PKC412 resistance in FLT3-mutant AML cells. Consistent with our previous studies of PKC412 treatment in conventional cultures of MM cells in isolation, we observed that PKC412 exhibits an anti-proliferative effect within the first 24 hrs of treatment, with major reduction of the numbers of viable cells at 48 and 72 hrs. At sub-micromolar doses that did not significantly affect the viability of non-malignant accessory cells tested, PKC412 had similar (or for some MM cell lines had more pronounced) activity against the MM cells, both in the presence and absence of the non-malignant accessory cells tested (HS-5, HS-27a, NIH-3T3 cells with or without transfection with human CD40L, etc.). In contrast, under the same experimental conditions, coculture with either BMSCs or exposure to their conditional media, decreased the response of MM cells to dexamethasone. These results suggested in contrast to the impact on FLT3mut AML, that the anti-MM activity of PKC412 is preserved (and in some cases slightly enhanced) when the MM cells interact with microenvironmental accessory cells and/or their secreted growth/survival factors. To obtain insight on possible mechanistic foundations of these observations, we examined the pattern of kinases inhibited by PKC412 at sub-μM concentrations (using FLT3 and FGFR3, known targets of PKC412 as positive controls). The results of in vitro kinase activity assays showed that PKC412 potently suppresses the aforementioned positive controls, but also exerts 〉50% inhibitory effect on the in vitro activity of additional kinases such as Akt2, Pim1, GSK3a, PDK1, p70S6K, SRC and Aurora A. Many of these kinases are known to participate in proliferative/anti-apoptotic signaling cascades downstream of cytokine/growth factor receptors or cell adhesion-mediated events triggered during MM–stromal interactions. We therefore conclude that the influence of the tumor microenvironment on the anti-neoplastic effects of PKC412 may be tumor-type dependent. The anti-MM activity of PKC412 is not subject to drug resistance triggered by non-malignant accessory cells, and conversely is occasionally moderately enhanced by these MM-stromal interactions. Mechanistically, this observation may be attributed in part to the multi-targeted nature of this inhibitor and, in particular, its aggregate impact on several kinases known to mediate stroma-induced proliferative and antipoptotic signaling in MM. Disclosures: Griffin: Novartis Pharmaceuticals: Consultancy, Research Funding. Richardson:Millennium: ; Celgene: ; Johnson & Johnson: ; Novartis: ; Bristol Myers Squibb:. Anderson:Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Mitsiades:EMD Serono: Research Funding; AVEO Pharma: Research Funding; Amgen: Research Funding; OSI Pharmaceuticals: Research Funding; PharmaMar: licensing royalties; Amnis Therapeutics: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.

Description: Extensive preclinical studies of several groups using tumor cells co-cultured with bone marrow stromal cells (BMSCs) has documented that the potent anti-MM activity of the proteasome inhibitor bortezomib is not suppressed by BMSCs (e.g. primary and immortalized BMSCs). Using our compartment-specific bioluminescence imaging (CS-BLI) assays, we extended these observations to larger panels of MM cell lines. We observed, however, a recurrent pattern that primary CD138+ MM tumor cells from bortezomib-refractory patients recurrently exhibited substantial in vitro response to clinically-achievable concentrations and durations of bortezomib treatment. To simulate this clinicopathological observation, MM.1R-Luc+ cells were injected i.v. in SCID-beige mice and treated with bortezomib (0.5 mg/kg s.c. twice weekly for 5 weeks): diffuse MM tumors initially responded to bortezomib, but eventually became refractory. These in vivo-resistant MM cells were isolated from the mice and were treated in vitro with bortezomib, exhibiteing similar responsinveness to this agent as their isogenic bortezomib-naive MM cells, To further address the possibility that this represents a previously underexplored form of a microenvironment-induced alteration in bortezomib responsiveness, we studied how MM cells respond to pharmacological proteasome inhibition after variable times of co-culture with BMSCs prior to bortezomib exposure. We observed that prolonged tumor-stromal co-culture (48-96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for many of the MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Notably, prolonged co-cultures with BMSCs prior to bortezomib treatment enhanced the activity of this agent for other MM cell lines (e.g. OPM1, Dox40, OCI-My5, KMS12BM or KMS18). However, MM.1S and MM.1R cells, when exposed to extended co-cultures with BMSCs prior to initiation of drug exposure, exhibited significant attenuation (2-3 fold increase of IC50 values) of their response to bortezomib in several independent replicate experiments. In support of these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells co-implanted with Luc-negative BMSCs did not show significant reduction in MM tumor growth with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.13), as quantified by bioluminescence imaging. In co-cultures with BMSCs, MM.1S and MM.1R cells also exhibited suppression of their response to carfilzomib (the degree of this stroma-induced resistance was more pronounced that in the case of bortezomib for these 2 cell lines). Consistent with these observations, in vivo administration of carfilzomib in the orthotopic model of diffuse bone lesions of MM.1R-Luc+ cells was associated with less pronounced reduction in tumor growth, compared to bortezomib treatment (p

Description: Abstract 1820 Proteasome inhibitors such as Bortezomib (Bort) represent a key drug class for the therapeutic management of multiple myeloma (MM). However, MM patients, even those who initially achieve complete clinical and biochemical remission with bortezomib-based regimens eventually relapse, and bortezomib-refractory disease is associated with short overall survival. Identifying the molecular basis of resistance to bortezomib is therefore crucial for the rational development of novel therapies to hopefully improve clinical outcome for advanced MM. To address this question, we generated an isogenic cell line model of bortezomib resistance by successive rounds of in vitro exposure of bortezomib-sensitive MM.1R cells to progressively increasing bortezomib concentrations. Serial dose-response analyses confirmed the generation of several clones with variable reduction in bortezomib-sensitivity (IC50 range 40–80nM vs.

Description: Abstract 1574 Cancer cells with stem cell-like features are a topic of intense research because their resistance to existing drugs is considered a culprit for relapses, even in patients with complete remission defined by clinical, biochemical and imaging parameters or by sensitive molecular techniques. Salinomycin, an antibacterial and coccidiodostatic ionophore, is reported (Cell 2009;138(4):645-59) to be 〉100-fold more potent against breast cancer cells with stem cell-like phenotype after mesenchymal transdifferentiation due to stable transfection with shRNA against CDH1 than against the parental cells. We evaluated whether salinomycin could also exhibit a similar activity against stem cell-like cells in multiple myeloma (MM). To establish a comparative reference for such potential activity, we first tested salinomycin (0-10 uM for up to 72hrs) against a panel of 15 MM cell lines and observed IC50 values 80% reduction of tumor cell viability in 6/15 cell lines tested at 0.5 uM, i.e. levels lower than the IC50 values for in vitro activity of salinomycin against breast cancer cells with (HMLE-shCDH1, IC50 ∼1 uM) or without (HMLE-shControl, IC50 〉〉10 uM) stem cell-like features. CD138+ purified primary tumor cells from 3 MM patients responded to salinomycin with IC50 values (105, 332 and 750 nM, respectively) in the same range as MM cell lines. In vitro combinations with bortezomib, doxorubicin, melphalan, and dexamethasone showed overall no antagonism, while evidence of additive or even synergistic effect could be identified in certain dose ranges. Because MM cell lines and primary tumor cells responded concordantly to salinomycin and with higher sensitivity than breast cancer stem cell-like cells, we hypothesized that MM cells may in general be more responsive to salinomycin than other tumors. Since tumor-stromal interactions can increase the expression of transcriptional signatures of “stemness” in MM cells, we embarked on characterizing the anti-MM properties of salinomycin using compartment-specific bioluminescence imaging (CSBLI) assays. These showed that co-culture with stromal cells did not confer resistance to salinomycin in 5 MM cell lines (MM.1S, OCI-My5, KMS-11, KMS-18, NCI-H929) and in fact enhanced its activity against 4 of them. Side population (SP) cells, defined by their ability to efflux Hoechst stain, represent a stem cell-like population which was identified in MM cell lines and could represent the functional equivalent of the mesenchymally transdifferentiated breast cancer stem cell-like cells. We observed that salinomycin reduces the SP fraction of MM cell lines at doses 〉20 times lower than those required for in vitro effect against the bulk of the respective cell lines. Interestingly, the anti-SP effect of salinomycin was more pronounced in the presence of stroma, similarly to the CSBLI studies on the entire MM cell population and consistent with our prior observation that tumor-stroma interaction enhances transcriptional signatures of ≪stemness≫ in the tumor compartment. However, when we tested the in vivo anti-MM activity of salinomycin in an orthotopic model of i.v. injected Luc+ MM cells, no anti-MM activity (in terms of tumor burden decrease or overall survival prolongation) was observed at the maximum tolerated dose (1 mg/kg i.p. daily, which is consistent with most studies reported thus far in the literature). Ex vivo treatment of KMS-11 cells with salinomycin doses (100 nM for 72 hrs) selectively targeting SP cells was followed by s.c. injection of these cells or vehicle-treated controls in sublethallly irradiated SCID/NOD mice, but no statistically significant improvement in tumor burden or overall survival was observed. Our in vitro results indicate that salinomycin exhibits intriguing in vitro anti-MM activity, not only against SP cells but also against the bulk ≪main≫ MM cell population, even in the presence of stromal support. In contrast, the in vivo activity of salinomycin is compromised by side effects in the orthotopic model of MM lesions, while short term ex vivo exposure of tumor cells is conceivably insufficient to eradicate clonogenic cells and lead to appreciable delay in tumor growth in vivo. Our studies point to intriguing features as well as notable challenges that have to overcome before salinomycin or other more selective agents of this class can be safely tested in clinical trials in MM. Disclosures: McMillin: Axios Biosciences: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mitsiades:Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.