Anticancer properties of bisaminoquinolines with modified linkers.

We have previously reported the unique features of dimeric bisaminoquinolines as anticancer agents and have identified their cellular target as PPT1, a protein palmitoyl-thioesterase. We now report a systematic study on the role of the linker in these constructs, both with respect to the distance between the heterocycles, the linker hydrophobicity and the methylation status (primary vs. secondary vs. tertiary) of the central nitrogen atom on the observed biological activity.

Divergent effects of acute and chronic PPT1 inhibition in melanoma.

Macroautophagy/autophagy-lysosome function promotes growth and survival of cancer cells, making them attractive targets for cancer therapy. One intriguing lysosomal target is PPT1 (palmitoyl-protein thioesterase 1). PPT1 inhibitors derived from chloroquine block autophagy, have significant antitumor activity in preclinical models and are being developed for clinical trials. However, the role of PPT1 in tumorigenesis remains poorly understood. Here we report that in melanoma cells, acute siRNA or pharmacological PPT1 inhibition led to increased ferroptosis sensitivity and significant loss of viability, whereas chronic PPT1 knockout using CRISPR-Cas9 produced blunted ferroptosis that led to sustained viability and growth. Each mode of PPT1 inhibition produced lysosome-autophagy inhibition but distinct proteomic changes, demonstrating the complexity of cellular adaptation mechanisms. To determine whether total genetic loss of Ppt1 would affect tumorigenesis in vivo, we developed a Ppt1 conditional knockout mouse model. We then crossed it into the BrafCA, PtenloxP, Tyr:CreERT2 melanoma mouse model to investigate the impact of Ppt1 loss on tumorigenesis. Loss of Ppt1 had no impact on melanoma histology, time to tumor initiation, or survival of tumor-bearing mice. These results suggest that chemical PPT1 inhibitors produce different adaptations than genetic PPT1 inhibition, and additional studies are warranted to fully understand the mechanism of chloroquine derivatives that target PPT1 in cancer.Abbreviations: 4-HT: 4-hydroxytamoxifen; BRAF: B-Raf proto-oncogene, serine/threonine kinase; cKO: conditional knockout; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9; DC661: A specific PPT1 inhibitor; DMSO: dimethyl sulfoxide; Dox; doxycycline hyclate; Easi-CRISPR: efficient additions with ssDNA inserts-CRISPR; GNS561/ezurpimtrostat: A PPT1 inhibitor; Hug: human guide; iCas: inducible CRISPR-Cas9; KO: knockout; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; LDLR: low density lipoprotein receptor; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NT: non-target; PTEN: phosphatase and tensin homolog; PPT1: palmitoyl-protein thioesterase 1; RSL3: RAS-selective lethal small molecule 3; SCRIB/SCRB1: scribble planar cell polarity protein; Tyr:CreERT2: tyrosinase-driven Cre recombinase fused with the tamoxifen-inducible mutant ligand binding domain of the human estrogen receptor; UGCG: UDP-glucose ceramide glucosyltransferase; WT: wild-type.

Targeting UGCG Overcomes Resistance to Lysosomal Autophagy Inhibition.

Lysosomal autophagy inhibition (LAI) with hydroxychloroquine or DC661 can enhance cancer therapy, but tumor regrowth is common. To elucidate LAI resistance, proteomics and immunoblotting demonstrated that LAI induced lipid metabolism enzymes in multiple cancer cell lines. Lipidomics showed that LAI increased cholesterol, sphingolipids, and glycosphingolipids. These changes were associated with striking levels of GM1+ membrane microdomains (GMM) in plasma membranes and lysosomes. Inhibition of cholesterol/sphingolipid metabolism proteins enhanced LAI cytotoxicity. Targeting UDP-glucose ceramide glucosyltransferase (UGCG) synergistically augmented LAI cytotoxicity. Although UGCG inhibition decreased LAI-induced GMM and augmented cell death, UGCG overexpression led to LAI resistance. Melanoma patients with high UGCG expression had significantly shorter disease-specific survival. The FDA-approved UGCG inhibitor eliglustat combined with LAI significantly inhibited tumor growth and improved survival in syngeneic tumors and a therapy-resistant patient-derived xenograft. These findings nominate UGCG as a new cancer target, and clinical trials testing UGCG inhibition in combination with LAI are warranted. SIGNIFICANCE: We discovered UGCG-dependent lipid remodeling drives resistance to LAI. Targeting UGCG with a drug approved for a lysosomal storage disorder enhanced LAI antitumor activity without toxicity. LAI and UGCG inhibition could be tested clinically in multiple cancers. This article is highlighted in the In This Issue feature, p. 247.

Lysosomal lipid peroxidation regulates tumor immunity.

Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

miR-196b-TLR7/8 Signaling Axis Regulates Innate Immune Signaling and Myeloid Maturation in DNMT3A-Mutant AML.

PURPOSE: DNMT3A mutations confer a poor prognosis in acute myeloid leukemia (AML), but the molecular mechanisms downstream of DNMT3A mutations in disease pathogenesis are not completely understood, limiting targeted therapeutic options. The role of miRNA in DNMT3A-mutant AML pathogenesis is understudied. EXPERIMENTAL DESIGN: DNA methylation and miRNA expression was evaluated in human AML patient samples and in Dnmt3a/Flt3-mutant AML mice. The treatment efficacy and molecular mechanisms of TLR7/8-directed therapies on DNMT3A-mutant AML were evaluated in vitro on human AML patient samples and in Dnmt3a/Flt3-mutant AML mice. RESULTS: miR-196b is hypomethylated and overexpressed in DNMT3A-mutant AML and is associated with poor patient outcome. miR-196b overexpression in DNMT3A-mutant AML is important to maintain an immature state and leukemic cell survival through repression of TLR signaling. The TLR7/8 agonist resiquimod induces dendritic cell-like differentiation with costimulatory molecule expression in DNMT3A-mutant AML cells and provides a survival benefit to Dnmt3a/Flt3-mutant AML mice. The small molecule bryostatin-1 augments resiquimod-mediated AML growth inhibition and differentiation. CONCLUSIONS: DNMT3A loss-of-function mutations cause miRNA locus-specific hypomethylation and overexpression important for mutant DNMT3A-mediated pathogenesis and clinical outcomes. Specifically, the overexpression of miR-196b in DNMT3A-mutant AML creates a novel therapeutic vulnerability by controlling sensitivity to TLR7/8-directed therapies.