Lactate dehydrogenase is crucial for tumor associated macrophage protection of multiple myeloma cells against chemotherapy Carolyn Stierhoff, Enguang Bi, Lintao Liu, Yong Lu, Xingzhe Ma, Jianfei Qian, Qiang Wang, Gang Xue, Maojie Yang, Tianshu Li, Qing Yi Multiple myeloma (MM) is a cancer characterized by an accumulation of malignant plasma cells in the bone marrow. Although chemotherapy is the most effective treatment, the majority of patients experience relapse. The major cause of treatment failure is the development of multidrug resistance. Thus, overcoming drug resistance will greatly improve patient survival. The enzyme lactate dehydrogenase (LDH) plays an important role in anaerobic respiration by converting pyruvate into lactate in the absence or low supply of oxygen. It has been observed that tumor- associated macrophages (TAMs) present in tumor environments can protect MM from chemotherapy treatment. We discovered that an LDH knockdown in myeloma cells decreased TAMs’ role in cell protection against chemotherapy treatment. Also, we found that, without LDH, macrophages were not able to differentiate into TAMs successfully. These results suggest that LDH activity in tumor environments promotes tumor survival, stimulates tumorigenesis, and manipulates local macrophages into performing pro-tumor functions. ABSTRACT CLEVELAND CLINIC LERNER RESEARCH INSTITUTE The standard treatment for MM is chemotherapy with combinations of drugs such as melphalan, thalidomide, bortezomib, and dexamethasone 1. Some cancer cells and tumors remain resistant or develop resistance to these treatments. TAMs have been shown to have both pro- and anti-tumor effects. Some of the anti-tumor effects include tumor angiogenesis, growth, development, and treatment resistance 2. In previous studies, it has been shown that, by affecting apoptotic signaling, TAMs protect myeloma cells with which they are in direct contact 3. The goal of this study was to explore possible mechanisms for myeloma cell protection by means of TAMs and their infiltration into myeloma tumor beds. Previously, other studies have associated high LDH levels with low prognosis and shortened survival rates for multiple myeloma patients 4. In order to explore the role of LDH in TAM-associated protection, a gene knockdown of LDH was performed. A knockdown involves a post-transcriptional silencing of a gene, in this case LDH, by the use of small RNA molecules which target gene transcripts for degradation, thus causing reduced expression of the target gene. In order to aid in development of more effective chemotherapeutic agents to combat MM, our study looks to identify the role of LDH in TAMs' protection of myeloma cancer cells through this knockdown method. INTRODUCTION RESULTS Figure 1. RT-PCR and western blotting showing that an LDH knockdown decreases LDH mRNA expression and detectable protein levels in myeloma cells. Figure 2. Flow cytometry using co-cultures of macrophage and myeloma cells representing increased myeloma cell death with melphalan treatment and LDH knockdown. Figure 3. RT-PCR showing the relative expression of the TAM markers ARG1 and VEGF. With LDH knockdown, ARG1 and VEGF expression is reduced. Figure 4. Flow cytometry showing a decrease in tumor- associated macrophage proliferation with the LDH knockdown. Methods RT-PCR and western blotting to determine the efficiency of the LDH knockdown. Flow cytometry with an apoptotic signaling assay to determine knockdown effects in myeloma/macrophage co-cultures. RT-PCR to identify the expression patterns of known TAM markers with an LDH knockdown. Flow Cytometry with a cell viability assay to determine TAM proliferation with an LDH knockdown. CONCLUSIONS An LDH knockdown decreases macrophages’ ability to protect myeloma cells from melphalan treatment. LDH knockdown decreases the expression of known tumor- associated macrophage markers. Knocking down LDH in myeloma prevents tumor-associated macrophages from growing and proliferating. REFERENCES 1 Multiple Myeloma. American Cancer Society, Jan Web. 8 March 8, Quatromoni, Jon G, and Evgeniy Eruslanov. “Tumor-Associated Macrophages: Function, Phenotype, and Link to Prognosis in Human Lung Cancer.” American Journal of Translational Research 4.4 (2012): 376–389. Print. 3 Zheng, Yuhuan et al. “Macrophages Are an Abundant Component of Myeloma Microenvironment and Protect Myeloma Cells from Chemotherapy Drug–induced Apoptosis.” Blood (2009): 3625–3628. PMC. Web. 8 Mar Evangelos Terpos et al. “High serum lactate dehydrogenase adds prognostic value to the international myeloma staging system even in the era of novel agents.” Eur J Haematol. 2010;85:114–119. PMC. Web. 8 March ACKNOWLEDGEMENTS Thank you to the Yi Lab and Cleveland Clinic Lerner Research Institute