PHF19 inhibition as a therapeutic target in multiple myeloma

Abstract

Epigenetic deregulation is increasingly recognized as a contributing pathological factor in multiple myeloma (MM). In particular tri-methylation of H3 lysine 27 (H3K27me3), which is catalyzed by PHD finger protein 19 (PHF19), a subunit of the Polycomb Repressive Complex 2 (PRC2), has recently shown to be a crucial mediator of MM tumorigenicity. Overexpression of PHF19 in MM has been associated with worse clinical outcome. Yet, while there is mounting evidence that PHF19 overexpression plays a crucial role in MM carcinogenesis downstream mechanisms remain to be elucidated. In the current study we use a functional knock down (KD) of PHF19 to investigate the biological role of PHF19 and show that PHF19KD leads to decreased tumor growth in vitro and in vivo. Expression of major cancer players such as bcl2, myc and EGR1 were decreased upon PHF19KD further underscoring the role of PHF19 in MM biology. Additionally, our results highlighted the prognostic impact of PHF19 overexpression, which was significantly associated with worse survival. Overall, our study underscores the premise that targeting the PHF19-PRC2 complex would open up avenues for novel MM therapies.

Figure 1.

Patients with high PHF19 expression (≥10.46) have significant worse overall survival compared to patients with low PHF19 expression (<10.46) shown here in the training set, A, and validation set B.

Figure 2.

Colony formation assay shows significant reduction in number of colonies in JJN3 and ARP1 cells with PHF19 KD compared to scrambled control (CTL), A+B, p<0.05. Furthermore, the colony sizes are both substantially reduced in both cell lines carrying the PHF19 KD compared to the control as seen under light- and fluorescent microscopy, C+D.

Figure 3.

Schematic overview of the xenograft model using the JJN3 cell line carrying either the PHF19 KD plasmid (n=10) or the CTL plasmid (n=10), A. Chemiluminescene imaging of 5 exemplary mice in the CTL vs PHF19KD group show more rapid tumor growth in the control group, B. Kaplan-Meier analysis shows that overall survival is worse in the mice with the CTL plasmid compared to the PHF19KD plasmid, C, p=0.05. Tumor burden measured by Kappa light chain (mg/dL) was significantly less in the PHF19 KD group compared to the control group, D,*= p<0.05.

Figure 4.

PHF19 KD lead to a decrease of H3K27me3 compared to scrambled CTL. The EZH inhibitor (Tazemetostat) at a concentration of 1μM was used as a negative control, A. A total of overlapping 228 genes were up- or downregulated by PHF19 KD in both cell lines (JJN3 and ARP1) ingenuity pathway analysis (IPA) elucidated pathways that were significantly affected by dysregulation of these overlapping genes after PHF19 KD, B p< 0.0001. Expression analysis of these overlapping 228 genes in the JJN3, C, and ARP1 cell lines, D, shows that several genes that are crucial to MM pathophysiology are affected by PHF19 KD, including bcl2, myc, egr1 and slam7. Genes with their corresponding proteins that are up- or downregulated in the same direction after PHF19KD, E.