MUC1-C Inhibition Leads to Decrease in PD-L1 Levels Via up-Regulation of Micro RNAs

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 co-stimulatory 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 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 down regulate the expression of PD-L1 protein in a lung cancer model. Indeed, silencing of MUC-1 in AML cell lines MOLM-14 and THP-1 lead to a marked increase in miR200c and miR34a levels. Moreover, ectopic overexpression of these miRNAs 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%) of miRNAs probed, of which 340 (42.4%) reached Bonferroni-corrected significance. Concordantly, MUC1 silenced THP-1 cells showed an increase in 698/801 (87.1%) of miRNAs probed, of which 154 (19.2%) reached Bonferroni-corrected significance, suggesting that MUC1 silencing resulted in a global increase in miRNA expression. 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. Immunoblotting of MUC1 silenced MOLM14 and THP-1 demonstrated an increase c-JUN expression, and moreover, inhibition of cJUN activity in AML cells lines using a peptide inhibitor of c-JUN, abrogated DICER expression in a dose dependent manner.

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.