Immunomodulatory Effect of MUC1-C in Acute Myeloid Leukemia
Authors: Stroopinsky Dina , Rajabi Hasan , Coll Maxwell Douglas , Pyzer Athalia Rachel , Rosenblatt Jacalyn , Jain Salvia , Luptakova Katarina , McMasters Malgorzata , Levine James D. , Joyce Robin , Arnason Jon E. , Karp Leaf Rebecca , Nahas Myrna Rita , Palmer Kristen Anna , Bar-Natan Michal , Tagde Ashujit , Washington Abigail , Somaiya Poorvi , Kufe Donald , Avigan David 
Published: October 2019

Acute myeloid leukemia (AML) is characterized by an immunosuppressive milieu in the bone marrow microenvironment that fosters tolerance, immune escape, and disease growth. Upregulation of PD-L1 expression by leukemia cells induces an exhausted phenotype in tumor reactive lymphocytes that blocks the activation and expansion of leukemia specific lymphocytes. Identifying pathways that regulate PD-L1 expression is vital for the development of novel therapeutic targets for immune based therapies. MUC1 is an oncoprotein that is aberrantly expressed on a majority of primary AML. MUC1 contains a transmembrane subunit (MUC1-C) that forms dimers and translocates to the nucleus where it mediates downstream signaling. MUC1-C expression on malignant cells promotes cell proliferation and resistance to apoptosis.

We have demonstrated that MUC1-C is a critical regulator of PD-L1 expression on AML cells. Silencing of MUC1 expression was documented following lentiviral transduction with MUC1-C specific shRNA using western blotting. MUC1 silencing results in the near abrogation of MUC1-C expression by the human AML cell lines, MOLM-14 and THP1. Silencing of MUC1-C on MOLM-14 and THP1 AML cells resulted in a two-fold increase in susceptibility to T cell mediated lysis as determined by a flourochrome based CTL assay.

To assess the effect of MUC1-C silencing on PD-L1 expression in-vivo, C57BL/6J mice were challenged with 100,000 GFP transfected TIB-49 murine AML cells in which MUC1-C was silenced using a lentiviral shRNA hairpin against MUC1-C. Following leukemia establishment, TIB-49 GFP+ cells were isolated from the bone marrows and spleens and PD-L1 expression on leukemic cells was measured. TIB-49 GFP+ cells of mice engrafted with MUC1 silenced AML cells demonstrated significantly lower PD-L1 expression compared to mice inoculated with TIB-49 cells transduced with a control vector (18% versus 3%; n=4). T cells isolated from bone marrow of mice inoculated with AML with silenced MUC1-C demonstrated a three-fold increase in INF-γ production when stimulated ex-vivo with autologous tumor lysate as compared to T cells from mice inoculated with control AML cells (n=4). Our group has developed a peptide inhibitor drug (G0-203), a cell-penetrating peptide that binds to the MUC1-C CQC motif, disrupting MUC1-C interaction with downstream effectors. As was observed following MUC1 silencing, bone marrow derived CD4+ and CD8+ T cells isolated from mice treated with G0-203 were shown to have two-fold increased intracellular IFN-γ production compared to control mice following ex-vivo exposure to AML lysate (n=4).

To investigate the mechanism by which MUC1-C regulates PD-L1 expression we examined the role of noncoding RNAs that have been shown to be effectors of oncogenic modulation. MicroRNAs (miRNAs) are a conserved class of small RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) of target mRNAs. The 3'UTR of the PD-L1 gene contains putative binding sites for miR-200 family of micro-RNAs, supporting the hypothesis that miR-200 plays a role in regulating the expression of PD-L1.

In the present study, we evaluated the effect of targeting MUC1-C mediated signaling, on miR200c levels and PD-L1 expression. MUC1-C silenced MOLM-14 cells demonstrated a two-fold increase in miR-200c expression, as demonstrated by qPCR and was associated with a reduction of PD-L1 expression from 77% to 13%. These data were confirmed in THP1 cells with PDL-1 expression decreasing from 95% to 40% following MUC1-C silencing. In support of the observation that miR200c is involved in the regulation of PD-L1 expression, lentiviral overexpression of miR200c in MOLM-14 cells led to a decrease in PD-L1 expression from 90% to 2% as demonstrated by flow cytometric analysis.

In conclusion, these results demonstrate that MUC1-C mediated signaling regulates the expression of PD-L1 on AML cells. Silencing MUC1-C results in reduced expression of PD-L1, interfering with immune escape and enhancing the expansion of leukemia reactive T cells. We define a novel mechanism by which MUC1-C governs PD-L1 expression through its modulation of miR200c. As such, MUC1-C functions as a critical regulator of immune escape and represents a novel target for immunotherapy.

Disclosures

Kufe: Genus Oncology: Consultancy, Equity Ownership.