Novel Protein Interactions Provide Insight into the Regulation of the Polymerase Associated Factor Complex in Acute Myeloid Leukemia
Authors: Ropa James , Serio Justin , Chen Lili , Chen Wei , Mysliwski Maria , Mellacheruvu Dattatreya , Basrur Venkatesha , Nesvizhskii Alexey , Muntean Andrew 
Published: November 2023

Epigenetic modifying proteins have been implicated in many forms of cancer including acute myeloid leukemia (AML). The Polymerase Associated Factor complex (PAFc) is an epigenetic regulator complex linked with the deposition of epigenetic modifications associated with transcriptional activation. The PAFc is composed of six subunits in humans‐ PAF1, CDC73, CTR9, LEO1, WDR61 and RTF1‐ many of which are linked with a variety of cancers including AML. We have previously shown that the PAFc is essential for AML cells that harbor chromosomal translocations involving the Mixed Lineage Leukemia gene (MLL1). The PAFc directly binds and recruits the resultant MLL‐fusion proteins as well as the wild type MLL1 histone H3K4 methyltransferase to target genes. This interaction drives a pro‐leukemic gene program that promotes self‐renewal and inhibits differentiation. Further, forced expression of the PAFc impedes myeloid differentiation, consistent with pro‐oncogenic functions. These data point to mechanisms regulating the PAFc function to facilitate hematopoietic differentiation and that may be circumvented in leukemic cells. The objective of our study is to identify and characterize biochemical mechanisms that regulate the PAFc function in normal and leukemic cells. Our methodology includes: 1) the analysis of cellular proliferation in the presence of wild type and mutated CDC73 proteins following the conditional knock out of Cdc73, 2) affinity purification‐mass spectrometry (AP‐MS) to identify novel PAFc interactions in AML cells and 3) mouse bone marrow colony forming assays to assess the function of novel PAFc interacting proteins on leukemic phenotypes. To investigate the regulation of the PAFc in AML, we generated MLL‐fusion driven leukemic cell lines from Cdc73 floxed bone marrow cells expressing a 4OHT inducible Cre. We demonstrated that loss of Cdc73 reduces proliferation of several subtypes of AML cells that rely on diverse molecular mechanisms for transformation. To investigate PAFc protein interactions, we generated a mutant (CDC73m) that has pro‐oncogenic functions in gastric cancer due to enhanced interactions with β‐Catenin. While wild type CDC73 rescued leukemic cell growth, CDC73m expression did not, suggesting context dependent functions of CDC73 and the PAFc in leukemic cells. Co‐immunoprecipitations demonstrate that CDC73m maintains interactions with the PAFc, MLL and β‐Catenin, suggesting this mutant is structurally intact. We hypothesized that CDC73m has an altered interactome compared to CDC73. We therefore performed AP‐MS and identified the H3K9 methyltransferases Setdb1 and G9a associated with CDC73m. Overexpression of SETDB1 or G9a reduced MLL‐fusion mediated colony formation and induced differentiation, consistent with a tumor suppressor function. Further, differentiation of human leukemic cells results in enhanced G9a interaction with CDC73. These data point to a role for temporal engagement of H3K9 methyltransferases with the PAFc to modulate gene expression that may relate to hematopoietic differentiation and transformation.