Addiction to Small Gtpase ARF6-Mediated Sphingolipid Homeostasis By AML but Not the Host
Authors: Zhao Helong Gary Gary , Johnson Kent , Pomicter Tony , Bates Briana , Bateman Benjamin , Ahmann Jonathan , Bishop Jenna , Yan Dongqing , Lee Greg , Haferlach Torsten , Zhu Weiquan , Odelberg Shannon , Deininger Michael 
Published: December 2023

Background
Cancer cells' unique metabolic demand has emerged as a promising therapeutic target in Acute Myeloid Leukemia (AML). The vesicular trafficking and signaling gatekeeper, ADP-Ribosylation Factor 6 (ARF6), belongs to the RAS superfamily of small GTPases and has been shown to regulate the homeostasis of lipids such as PIP2, phosphatidic acids, and cholesterol. Here we describe AML cells' unusual addiction to ARF6-mediated sphingolipid homeostasis and its clinical significance.
Experiments and Results
ARF6 knockdown (KD) or CRISPR-mediated ARF6 knockout (KO) inhibited the proliferation of AML cell lines, THP-1 and MV4-11, as well as the colony formation of primary AML CD34 + cells. Surprisingly, Tamoxifen-induced whole body KO of Arf6 in adult Arf6 f/f;Rosa-CreERT 2 mice led to healthy and grossly normal mice with a normal lifespan. Adult mice lacking ARF6 show no abnormality in complete blood count, urine test, or histology. Hence, ARF6 is genetically indispensable for AML, but not for the host. To confirm AML's addition to ARF6 in vivo, we introduced Arf6 KO in an MLL-AF9-induced AML model in C57BL6/J mice. We are currently following up with the disease progression between Arf6-KO AML group and control AML group, and expect to obtain results before the 2023 ASH meeting.
After discovering that ARF6 is genetically necessary for AML but not needed for the host, we analyzed its clinical significance. Since ARF6 mutations are not detected in AML samples, we focused on analyzing its expression. With univariate analysis, we show that high ARF6 expression (top 50%) is significantly associated with worse overall survival (OS) and worse disease-free survival (DFS) of AML patients in the TCGA-NEJM-2013 (TCGA) cohort (N=200) and the Münchner Leukämielabor (MLL) cohort (N=579). In multi-variate analysis, high ARF6 expression is significantly associated with worse OS in the BeatAML cohort (N=562) and shows a trend towards worse OS and DFS in the TCGA cohort.
Following a hypothesis-driven approach, we investigated the role of ARF6 in regulating sphingolipid homeostasis by HPLC-MS lipidomic analyses of AML cells (THP-1) with or without ARF6 depletion. Lipidomic profiles revealed that ARF6 KD led to an increase of the overall ceramide/sphingomyelin ratio, suggesting a defect in sphingolipid homeostasis. We then analyzed the expression of sphingolipid metabolic enzymes and observed that ARF6 KD downregulated sphingomyelin synthase 1 and 2 (SGMS1 and SGMS2) expression, hindering ceramide-to-sphingomyelin conversion. Supporting this, we found a positive linear correlation between ARF6 and SGMS1/2 expression among the patients in TCGA cohort, confirming their correlation. High expression of the ARF6 effectors SGMS1/2 is also associated with worse OS and DFS in the TCGA cohort and the MLL cohort in univariate analysis, resembling that of ARF6. Downstream of ceramide accumulation, ARF6 KD inhibited the pro-survival AKT pathway, confirming the pro-apoptotic roles of ceramide in cancers. The anti-proliferative effect of ARF6 depletion can be rescued by sphingomyelinase inhibitor GW4869 (blocking ceramide production) or by expressing a constitutively active form of AKT (AKT1 E17K).
To pharmacologically target ARF6, we developed two generations of ARF6 small molecular inhibitor pro-drugs, named NAV-2729 and A6-4471. NAV-2729 and A6-4471 showed high specificity to ARF6 without inhibiting other small GTPases, with in vitro IC 50 in the lower μM range. Both NAV-2729 and A6-4471 inhibited AML cell line proliferation and reduced primary AML cell colony formation in lower μM range. Cord blood normal CD34 + cells are not sensitive to ARF6 inhibitors, but non-specific toxicity was observed at higher concentrations. When tested in vivo with THP-1 xenografts in NSG mice, both inhibitors reduced circulating AML cells without obvious toxicity to the host.
Conclusion
ARF6 is necessary for AML proliferation and its higher expression is associated with worse patient survival. However, ARF6 is dispensable for normal host physiology. Mechanistically, ARF6 maintains critical ceramide/sphingomyelin balance in AML by regulating SGMS1/2 expression. Pharmacologically inhibiting ARF6 suppressed AML proliferation in vitro and in vivo in a genotype-agnostic fashion. Our study implicates ARF6 as a potentially actionable and safe target in AML.