Background: Cancer stem cells (CSCs), a minor subset of cancer cells, mediate aggressiveness, metastasis, and drug resistance of many cancers including the deadly pancreatic cancer (PC). Molecular governance of pancreatic CSCs remains elusive, mandating the need for their better molecular characterization to improve management of PC. PD2 (Pancreatic Differentiation 2) or RNA Polymerase II-Associated Factor 1 (Paf1) is a core component of human PAF1 complex (PAF1C), which regulates transcription elongation and mRNA processing. Human PAF1C consists of 5 members: PAF1, LEO1, CDC73, CTR9 and SKI8. Recently, PAF1 has emerged as a novel pancreatic CSC marker that enhances tumorigenic and metastatic potential of PC. Paf1 also maintains the self-renewal of mouse embryonic stem cells and ovarian CSCs via its interaction with OCT3/4, a major regulator of pluripotency. However, the mechanistic role of PAF1 in CSC maintenance and CSC-mediated PC pathogenesis is poorly understood.

Hypothesis: We hypothesize that “PAF1 forms a sub-complex exclusive of PAF1C, wherein PAF1 functions as the master-regulator for maintaining pancreatic CSCs by regulating stem cell gene signature”.

Experimental Design: PAF1 knockdown (KD) was used to identify its targets and role in tumorigenesis. PAF1 sub-complex in CSCs was identified using immunoprecipitation (IP) and mass spectrometry (MS). ChIP-Seq was performed to confirm binding of PAF1 on its target genes.

Results: Inducible KD of PAF1 led to a significant reduction in tumor burden from orthotopically implanted human PC cells in athymic nude mice, indicating its role in pancreatic tumorigenesis. CSCs from different PC cell lines demonstrated higher expression of PAF1 along with CSC markers and PAF1 KD caused a significant decrease in CSC and self-renewal markers analyzed through Western blotting and immunofluorescence. Reciprocal co-IP and MS revealed that PAF1 interacted with Phf5a, DDX3, and hnRNP-K in CSCs. PAF1 KD in CSCs showed significant down-regulation of tumorigenic and stemness maintenance genes by RNA-Seq and PCR array analysis. PAF1 along with its binding partners occupied Nanog promoter in pancreatic CSCs. Further, KD of PAF1 in CSCs did not affect the expression of other PAF1C components, whereas individual KD of single PAF1C components decreased other remaining PAF1C members including PAF1. Moreover, other PAF1C components did not interact with Phf5a, a PHD-fold harboring nuclear protein in CSCs, suggesting that PAF1 forms a sub-complex with Phf5a, independent of PAF1C that functions in CSC maintenance. Depletion of Paf1 from mouse pancreas since birth using a CRISPR/Cas9-based conditional Paf1 knockout mouse model severely affected exocrine pancreas development with peri-ductal sclerosis and inflammation.

Conclusion: Altogether, PAF1 functions as the master-regulator for CSC maintenance by forming a sub-complex, which regulates CSC-network genes in PC.

Citation Format: Saswati Karmakar, Sanchita Rauth, Rama Krishna Nimmakayala, Srikanth Barkeer, Mohd W. Nasser, Satyanarayana Rachagani, Dario Ghersi, Moorthy P. Ponnusamy, Surinder K. Batra. A novel mechanism for pancreatic cancer stem cell maintenance: PAF1C-independent role of PAF1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3679.