Program: Oral and Poster Abstracts
Type: Oral
Session: 201. Granulocytes, Monocytes and Macrophages I
The murine AML cell line TIB-49 was transplanted in C57BL/6J mice. Following establishment of disease, the mice were euthanized, along with healthy controls, and bone marrow and spleen CD11b+ Gr1+ MDSCs were quantified by flow cytometry. Engrafted mice had an average MDSC burden of 47% in the marrow and 8.7% in the spleen, compared with 35% and 2% in control mice, respectively. The increase in MDSCs was granulocyte predominant, consistent with our findings in patients with AML. Gr1+CD11b+ cells derived from the marrow and spleens of engrafted mice showed higher levels of Arginase-1 compared to MDSCs from control mice, suggesting an increase in immune suppressive phenotype.
To investigate the role of MUC1 on the expansion of MDSCs, expression of MUC-1C, the signaling C-terminus of MUC1, was silenced in TIB-49 cells by stable expression of a MUC1-C shRNA as determined by Western Blot analysis. MUC1 silenced or control vector transduced TIB-49 cells were transplanted into C57BL/6J mice. Following establishment of disease, the mice were euthanized, and bone marrow and spleens were quantified for MDSCs. Control AML engrafted mice had an average splenic MDSC burden 2-fold higher than MUC1 silenced AML engrafted mice (n=4). We have developed a cell-penetrating peptide (GO-203) that disrupts homodimerization of the MUC1-C subunit necessary for its downstream signaling. C57BL/6J mice were challenged with TIB-49 AML cells and after 24 hours were treated daily with GO-203. Mice treated with the MUC1 inhibitor had a 2-fold decrease in splenic MDSCs, compared to control mice (n=3) at time of analysis following disease establishment.
Noncoding RNAs have emerged as a critical biologic effector of oncogenic pathways. MicroRNAs (miRNAs) post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. miR-34a has been implicated in regulating the expansion of MDSCs. In the present study we demonstrated that silencing of MUC1 expression via lentiviral transduction with a MUC1 specific shRNA resulted in a significant increase in miR-34a expression, as quantified by q-PCR. Consistent with the MUC1 mediated regulation of MDSC expansion by modulation of miR-34a levels, MDSCs induced by co-culture of healthy donor PBMCs with MUC1 silenced AML cells contained 4-fold higher levels of miR-34a, as compared to controls. AML cells were pre-incubated with SYTO® RNASelect™ Green Fluorescent cell stain. After 4 hours, co-cultures of PBMCs and AML cells were analyzed via flow cytometry. AML cells were excluded and cells positive for MDSC markers and containing green fluorescing exosome dye were quantified. AML derived exosomes were found in 18% of MDSCs expanded from AML cells (n=3), demonstrating exosome trafficking from tumor to MDSCs. Finally, miR-34a was over-expressed in MOLM-14 using lentiviral transduction. Over-expression of miR-34a in MUC1 expressing MOLM-14 cells resulted in a 30% reduction in MDSC expansion in co-cultured PBMCs (n=3).
In conclusion, MUC1 regulates MDSC expansion in AML, via its effects on miR34a, acting as a critical mediator of tumor mediated immune suppression. Incorporating strategies to reverse the expansion of MDSCs in AML, potentially by targeting MUC1 and increasing miR-34a expression offers a novel therapeutic approach for cancer immunotherapy.
Disclosures: Küfe: Genus Oncology, LLC: Equity Ownership . Avigan: Regimmune: Research Funding .
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