Bone Marrow Microenvironment Induces Genomic Instability and Enables Clonal Evolution in Multiple Myeloma

Abstract

Multiple myeloma (MM) is characterized by ongoing DNA damage and genomic instability, with acquisition of mutations and structural changes during disease progression. Its growth and survival charactersitics are also affected by the bone marrow (BM) microenvironment, influencing its pathogenesis as well as development of drug resistance. However, it is unclear whether the BM microenvironment can affect the ability of the tumor cells to acquire new genomic changes. The susceptibility to genomic changes might come from an increase in the endogenous sources of DNA damage or an impairment or deregulation of the DNA Damage Response (DDR). We hypothesized that the microenvironment may also contribute to ongoing DNA damage and genomic instability in MM.To investigate the potential role of the microenvironment in genomic stability in MM, we evaluated change in frequency of spontaneous DNA damage in myeloma cells when co-cultured with various microenvironment cells (HS-5 stromal cell line and primary patient BM stromal cells [BMSC]). We observed that DNA breaks, already present at baseline as demonstrated by high levels of pH2AX, were significantly increased when MM cell lines were co-cultured with either HS5 cell or primary BMSCs (2-4 folds increase, p <0.05). Moreover, similar results were observed when MM cell lines were cultured in presence of stromal cell supernatant or IL6 alone, also suggesting a possible effect of the cytokine. Immunofluorescence experiments revealed that after co-culture with stromal cells pH2AX recruitment to sites of DNA DSBs was also increased.Since we have shown that elevated HR and nuclease (especially apurinic/apyrimidinic endonuclease) activities are important mechanisms of genomic instability in MM, we next evaluated the impact of microenvironment on nuclease and HR activities using functional assays. Interestingly, we found that MM-BMSC co-culture induced a significant increase in overall nuclease activity, with specific increase in the expression of APEX1, a major apurinic/apyrimidinic (AP) nuclease, leading to increase in AP acitivity as measured by a flourescent-based oliginucleotide assay. MM-BMSC co-culture also induced HR activity demonstrating the impact of BMSC on genomic instability.To more comprehensively evaluate effects of stromal cells on different DNA Damage Response pathways, we performed RNA-sequencing of 3 different MM cell lines cultured alone or in presence of HS-5. We observed that the network of genes involved in DNA Damage Response were significantly altered in MM in the presence of stromal cells. We further confirmed our results in publicly available microarray gene expression data in MM cell lines cultured alone or with HS-5. These results support the idea that microenvironment can impact DNA integrity and stability in MM cells.Finally, we cultured MM cell lines alone or in presence of BMSC from MM patients for 4 weeks and investigated genomic changes using cytoscan HD array to detect effects of co-culture on acquisition of new copy-number alterations. We observed that MM cell line growth in the presence of BMSCs provides a selective environment for clonal selection, demonstrating the role of stromal cells in clonal diversity in MM.In conclusion, our data suggest that BMSC enhance DNA damage and instability in MM. Understanding the mechanisms leading to genomic instability is important to develop new therapeutic strategies aimed at preventing MM evolution. DisclosuresAnderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder.