P-402: Inhibition of proline production by stromal cells negatively impacts myeloma tumor growth by reducing cytokine and growth factor secretion.

Introduction: The bone marrow microenvironment plays a crucial role in the development of drug resistance in multiple myeloma (MM). In recent years, metabolomics have become of great importance in the search for new treatment strategies in cancer. Cancer cells are highly capable of adapting their metabolism to increasing energy demands. We have previously shown that MM cells are dependent on proline synthesis, and inhibition of proline production by PYCR1 interference increased bortezomib sensitivity both in vitro and in vivo. Surrounding stromal cells in the microenvironment support tumor growth. As PYCR1 is highly expressed in stromal cells, we wondered whether the stromal cells produce and secrete proline into the microenvironment to support and stimulate MM cell metabolism and proliferation. In this study, we investigated whether inhibition of proline production by stromal cells negatively affects myeloma cell viability.

Methods: PYCR1 expression was investigated by the use of microarray data from the Heidelberg/Montpellier cohort. For in vitro experiments, the human MM cell lines OPM-2 and XG-2 and the human stromal cell line HS-5 were used. Data was confirmed on primary CD138+ MM cells. To obtain primary stromal cells, the CD138- fraction from primary patient samples was plated out in fresh medium. After 48h, the medium was refreshed and all adherent cells were further cultured and used as primary stromal cells. PYCR1 expression in HS-5 cells was reduced through siRNA. Hypoxic culture ( < 1% O2) was established through hypoxia chambers. Angiogenesis and cytokine arrays were performed to identify changes in cytokine and growth factor secretion. Protein expression was measured by western blot. Viability was assessed by CellTiterGlo assay.

Results: Gene expression analysis showed high RNA expression of PYCR1 in healthy BMPCs, plasma cells from MGUS and MM patients, but also in bone marrow stromal cells (BMSCs). PYCR1 expression was low or absent in other cell types, including T cells, osteoclasts, monocytic and granulocytic cells. On protein level, we also confirmed PYCR1 expression in MM cells (CD138+), the CD138- fraction and cultured primary stromal cells. Moreover, PYCR1 expression increased upon hypoxic culture in primary stromal cells and HS-5. PYCR1 knockdown in HS-5 did not affect the viability of the stromal cells, but did reduce their protective effect on OPM2 and XG-2 cells against bortezomib. Further analysis of the conditioned medium of the stromal cells via angiogenesis and cytokine arrays identified a reduced secretion of IL-1β, activin A and IL-13 upon PYCR1 interference. In vivo, PYCR1 inhibition enhanced bortezomib-mediated effects on tumor burden.

Conclusions: This study indicates that PYCR1 is a potential target in the MM tumor microenvironment, to not only alter metabolite availability for MM cells, but to also reduce cytokine and growth factor release from stromal cells, thereby annihilating their protective effect on MM cells.