The human fibroblast growth factor/fibroblast growth factor-receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenstrom's Macroglobulinemia (WM). Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We investigated the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule, NSC12, a small molecule identified using pharmacophore modeling of the interaction of a minimal PTX3-derived FGF-binding pentapeptide with FGF2.
By interrogating the transcriptome signature of patients' BM-derived CD19-positive cells (GEO9656, GEO6691), we found a significant enrichment of FGF/FGFR-driven signaling cascades, including PI3K-AKT, MAPK and STAT3 pathways (FDR<0.25; P<0.05); coupled with higher expression of FGFs (FGF2, FGF7, FGF12, FGF18; P<0.05) in WM cells as compared to their normal cellular counterpart. FGFRs are also shown to be overexpressed in WM. We performed transcriptome profiling of NSC12-treated WM cells, confirming the blockade of the FGFR-signaling blockade; and, importantly, we discovered the efficacious silencing of MYD88 and MYD88-dopwnstream target HCK in WM cells. These findings were confirmed at protein level, showing inhibition of MYD88-driven pathways, such as BTK-, and SYK-phosphorylation. As a further demonstration of the functional impact of NSC12-dependent targeting of MYD88, we could confirm the inhibition of both canonical and non-canonical NF-kB in NSC12-treated WM cells, as assessed at nuclear protein level by WB and by using the NFkB activity assay.
In addition, the NSC12-dependent inhibition of MYD88 resulted in silencing of the MAPK-ERK signaling cascade, thus leading to NSC12-induced Myc-silencing in WM cells. We next confirmed the efficacy of NSC12 in silencing bone marrow stromal cell (BMSC)-induced FGFR3 phosphorylation; paralleled by inhibition of of pro-survival pathways, including pAKT, the AKT-downstream pGSK3β; p-ERK; and p-STAT3. Functional sequelae of the FGF/FGFR blockade in WM cells were studied, demonstrating inhibition of WM cell growth, induction of apoptosis, enhanced ER stress and initiation of UPR. Of note, anti-WM activity of NSC12 was also documented using primary bone marrow-derived CD19+ cells isolated from patients with WM. In contrast, NSC12 did not show cytotoxicity on PBMC-derived CD19+ cells isolated from healthy donors.
The anti-WM activity exerted by NSC12 was confirmed also within the context of the supportive bone marrow milieu, as shown both in vitro and in vivo. BCWM.1, MWCL1 cells were cultured with NSC12 in the presence or absence of primary WM BMSCs: adherence of WM cells to BMSCs triggered a significant increase in the proliferation, which was inhibited by NSC12in a dose-dependent manner (P<0.05). Using a disseminated humanized WM model, BCWM.1 M-Cherry-Luc tumor-bearing mice were treated with either NSC12 or vehicle control. NSC12-treated mice presented with a significant inhibition of WM tumor growth as demonstrated by bioluminescence imaging analysis. NSC12-dependent reduction of WM tumor cell BM infiltration was confirmed by IHC performed on harvested femurs, in comparison to vehicle-treated mice. Importantly, we assessed MYD88-mRNA expression using harvested bone marrow femurs; and found a significant reduction of MYD88- and MYD88-downstream target HCK in NSC12-treated mice was confirmed ex vivo (P<0.05).
Overall, our studies are reporting on the use of NSC12, as a novel potential therapeutic strategy to specifically halt the FGF/FGFR axis in WM; and demonstrate how the observed anti-WM activity exerted by NSC12 may be driven, at least in part, by inhibition of MYD88.
Motta:Roche: Honoraria; Janssen: Honoraria. Rossi:Alexion: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Advisory board; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria; Amgen: Honoraria; Sanofi: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Roccaro:Amgen: Other; AstraZeneca: Research Funding; Celgene: Other; Janssen: Other; Italian Association for Cancer Research (AIRC): Research Funding; Transcan2-ERANET: Research Funding; European Hematology Association: Research Funding.
Asterisk with author names denotes non-ASH members.