Small Molecule Inhibition of PDE6D-RAS Interaction Suppresses the Growth of Acute Lymphoblastic Leukemia Cell Lines

Abstract

The activation of RAS signaling has been shown to act as the driver of both de novo and relapsed, chemotherapy resistant acute lymphoblastic leukemia (ALL). Full RAS transformation requires the activity of the small RAS-related C3 botulinum toxin substrate (RAC) protein family, including the hematopoietic-specific RAC2 GTPase and we have previously demonstrated the role of RAC in specific leukemia types. Even though relapsed ALL patients have a 34% overall prevalence of RAS-activating mutations, KRASG12C mutations were not present, suggesting that the only RAS inhibitor currently available (G12C-specific) would not be effective in treating these patients. Phosphodiester 6 subunit delta (PDE6D), initially identified as a subunit of rod-specific photoreceptor phosphodiesterase, is now also known as a transporter of prenylated cargo. In fact, PDE6D has been shown to modulate the activity of RAS family proteins by regulating their subcellular location. When active, RAS proteins migrate to the cell membrane where they interact with a number of effectors triggering pro-survival downstream pathways including the mitogen-activated protein kinase / extracellular signaling-regulated kinase (MAPK/ERK) and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT). MAPK/ERK and PI3K/AKT pathways in particular are believed to synergize to induce survival and cellular transformation. We carried out a biological screen of small molecular compounds that assessed the inhibition of RAS-mutated ALL cell lines' proliferation through MTT assays, inhibition of RAC activation and the absence of inhibition of normal hematopoietic progenitor growth in colony forming unit (CFU) assays. Lead compounds were further evaluated for their lipophilicity, solubility and potency of biological activity. Here we report the identification of DW0254 which demonstrates arrest of proliferation and induction of apoptosis in RAS-mutant human B- and T-ALL cell lines. We have identified PDE6D as the putative target of this compound through photoaffinity labeling mass spectroscopy (PAL-MS). The cocrystal structure of DW0254 with recombinant PDE6D demonstrated that this small molecule fits inside the hydrophobic pocket and forms hydrogen bond interactions with residues Q88, Y149 and R61. From a molecular perspective, the occupation of the pocket by DW0254 leads to decreased interaction between PDE6D and farnesylated RAS. This impairment of PDE6D's ability to transport RAS leads to the delocalization of both mutant-NRAS and -KRAS4B proteins from the cell membrane to the cytosol, confirmed by real-time fluorescent imaging of recombinant GFP-RAS proteins. Ultimately, RAS delocalization upon DW0254 binding to PDE6D leads to decreased activation of both MAPK/ERK and PI3K/AKT pathways, and potent inhibition of RAC GTPases. CRISPR Cas9 saturating mutagenesis experiments confirmed that mutations in the farnesyl binding pocket leads to compound resistance, giving direct evidence that leukemia growth arrest is caused by molecule binding to PDE6D. Cells that showed increased IC 50 to DW0254 after mutagenesis did not exhibit resistance to Deltarasin, a previously described PDE6D inhibitor. DW0254 anti-leukemic activity was confirmed in an ex vivo murine xenograft model using short-term treated human NRAS-mutated ALL cell line P12-ICHIKAWA. After transplant, DW0254 treated cells showed impaired tumorigenic and engraftment potential when compared to vehicle controls. In conclusion, we have identified DW0254, a PDE6D inhibitor that has anti-leukemic activity in RAS-mutated ALL cell lines. We have successfully co-crystalized this compound with PDE6D showing binding to its farnesyl binding pocket and confirmed that the mechanisms of action of this inhibitor involve the loss of membrane localization of RAS and consequent inhibition of signaling to its downstream effectors. Pocket mutations validate the hypothesis that the effects observed derive from the binding of DW0254 to PDE6D and not from off-target effects. Ex vivo experiments show promising anti-leukemic effects and set the basis for future compound optimization. Disclosures De Vita: Novartis: Current Employment. Anighoro: Relation Therapeutics: Current Employment; Evotec SAS: Ended employment in the past 24 months. Autelitano: Evotec SAS: Current Employment. Beaumont: Evotec SAS: Current Employment. Klingbeil: Evotec SAS: Current Employment. Ermann: Evotec SAS: Ended employment in the past 24 months. Williams: BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding.