Abstract 54: MUC1-C regulates PD-L1 expression in acute myeloid leukemia, via downregulation of miRNAs
Authors: Stroopinsky Dina , Pyzer Athalia Rachel , Anastasiadou Eleni , Rajabi Hasan , Washington Abigail , Tagde Ashujit , Coll Maxwell , Cole Leandra , Palmer Kristen , Somaiya Poorvi , Leaf Rebecca Karp , Nahas Myrna , Apel Arie , Jain Salvia , Joyce Robin , Arnason Jon , Slack Frank , Kufe Donald , Rosenblatt Jacalyn , Avigan David 
Published: August 2018

Abstract
Acute myeloid leukemia (AML) is a lethal hematologic cancer in which the tumor microenvironment is characterized by a state of immune dysregulation that fosters disease progression. The PD-L1/PD-1 pathway confers a negative costimulatory signal that induces T-cell exhaustion, supports immune evasion by malignant cells, and is a critical component of the immunosuppressive milieu in AML. While PD-L1 expression in AML is dynamic, little is known about the mechanisms responsible for regulating PD-L1 expression in AML. MUC1 is a heterodimeric oncoprotein aberrantly expressed in solid tumors and hematologic malignancies including AML, which supports critical aspects of the malignant phenotype including cell proliferation, self-renewal, and resistance to apoptosis. Given its critical function is supporting the malignant phenotype of AML cells and its presence on leukemia stem cells, we sought to explore its role in mediating the immunosuppressive milieu of the tumor microenvironment.
In the present study, we have demonstrated that suppression of MUC1-C expression via MUC1-specific shRNA or CRISPR/Cas9 gene editing in cell lines, or with a peptide inhibitor of MUC1 signaling (GO-203) in primary patient AML samples, results in the near abrogation of PD-L1 expression as shown by flow cytometry and immuno-blotting. Interestingly, no decrease in PD-L1 mRNA expression was detected by qPCR, suggesting a post-transcriptional regulation of PD-L1 expression. Noncoding RNAs have been shown to regulate critical aspects of oncogenic phenotype through the selective binding to target mRNAs and regulation of protein translation. The microRNAs miR200c and miR34a demonstrate homology with the 3'UTR section of PDL-1 mRNA. Mir200c was recently shown to downregulate the expression of PD-L1 protein in a lung cancer model. Indeed, inhibition of MUC-1 in AML cell lines and patient samples leads to a marked increase in miR200c and miR34a levels. Moreover, ectopic overexpression of these miRNAs in MOLM-14 and THP-1 resulted in near abrogation of PD-L1 expression. To examine if the increase in miR200c and miR34a was representative of a class effect on miRNAs, a miRNA NanoString array was performed in MUC1-silenced MOLM-14 and THP-1 cell lines. MUC1-silenced MOLM-14 cells showed an increase in 786/801 (98.1%) and 698/801 (87.1%) of miRNAs probed in the respective cell lines.
miRNAs are processed from precursor (pre-) miRNAs to mature miRNAs by the proteins DICER and Argonaut-2. To elucidate the mechanism of MUC1-C regulation of miRNA expression, MUC1-silenced MOLM-14 and THP-1 cells were evaluated for their expression of pre-miR200c and pre-miR34a. The results demonstrated no change in the miRNA precursor levels, in contrast to the levels of mature miRNAs. This suggested that MUC1-C might regulate the processing of precursor miRNAs to their mature form. MUC1-silenced MOLM-14 and THP-1 demonstrated an increase in the mRNA and protein expression of DICER, but not Argonaut-2. To elucidate the mechanism of MUC1-C regulation of DICER expression, we examined transcription factors known to regulate DICER. cJUN is a key regulator of DICER expression that has been reported to be repressed by MUC1 signaling.
MUC1-silenced MOLM-14 and THP-1 cells demonstrated an increase in c-JUN mRNA levels and protein expression. Furthermore, inhibition of cJUN activity in AML cell lines using a peptide inhibitor of c-JUN abrogated DICER expression and restored PD-L1 expression in a dose dependent manner. Moreover, immuno-blotting of MUC1-silenced MOLM-14 and THP-1 cells demonstrated increased expression of total and phosphorylated JNK and ERK, both well known to be mediators of cJUN phosphorylation and transcription, respectively.
In conclusion, these findings strongly suggest MUC1-C as a key regulator of microRNA expression and demonstrate a critical mechanism by which MUC1-C signaling exploits noncoding RNAs to promote PD-L1 expression, resulting in an immunosuppressive characteristic of AML cells.
Citation Format: Dina Stroopinsky, Athalia Rachel Pyzer, Eleni Anastasiadou, Hasan Rajabi, Abigail Washington, Ashujit Tagde, Maxwell Coll, Leandra Cole, Kristen Palmer, Poorvi Somaiya, Rebecca Karp Leaf, Myrna Nahas, Arie Apel, Salvia Jain, Robin Joyce, Jon Arnason, Frank Slack, Donald Kufe, Jacalyn Rosenblatt, David Avigan. MUC1-C regulates PD-L1 expression in acute myeloid leukemia, via downregulation of miRNAs [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 54.