Cells of the Osteoblast Lineage Confer Myeloma Cell Resistance to Established and Investigational Therapeutic Agents

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.

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.

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.

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.

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.