Nanoliposomal C6-Ceramide Exhibits Synergistic Anti-Cancer Activity with a Small Molecule Agonist of PP2A in Chronic Lymphocytic Leukemia

The introduction of targeted therapies, including venetoclax (inhibitor of Bcl-2) and ibrutinib (inhibitor of Bruton's tyrosine kinase), has significantly advanced treatment of non-Hodgkin's lymphoma, particularly Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). However, patients treated with ibrutinib, venetoclax, or the combination of ibrutinib and venetoclax are likely to exhibit treatment relapse. We have previously published that microenvironmental factors can generate anti-apoptotic resistance to combinatorial ibrutinib/venetoclax in both CLL and MCL models via NF-kB-dependent upregulation of anti-apoptotic proteins (Mcl-1 and Bcl-xL) (Jayappa et al. 2017). We have recently published the discovery of phenotypically similar drug resistant CLL cells that overexpress multiple anti-apoptotic proteins in patients de novo. These cells were enriched during treatment with venetoclax, suggesting this pool of resistant persister cells could be a source of treatment failure (Jayappa et al. 2021). We have previously formulated a non-toxic C6-ceramide nanoliposome (CNL) that greatly improves bioavailability of this bioactive sphingolipid and exerts anti-cancer activity in vivo, including CLL cell line that exhibits multidrug resistance phenotype (Lindsay et al. 2013). Thus, CNL-based novel therapies could hold promise for depleting the pool of multidrug resistant cancer cells in patients.

In the current study, we screened CNL activity in CLL patient PBMC samples (N=15) using a flow cytometry-based cytotoxicity assay ex vivo. Our analysis revealed that CNL induced apoptotic/non-apoptotic cell death at overly high concentrations (EC₅₀, 31 µM), prompting us to search for potential combinatorial therapies with enhanced toxicity profiles. To address this, we performed a combination drug screen with CNL as an anchor agent in a multidrug resistant CLL cell line (MEC1, p53^-/mt) using a cell survival assay. Our analysis discovered exceptional synergistic interaction between CNL and a small molecule activator of Protein Phosphatase 2A (PP2A) (SMAP, DT061) in MEC1 cells [Bliss score (actual-Bliss predicted cytotoxicity): 56% (CNL, 6 µM; DT061, 12 µM)] and this synergistic behavior was further validated in primary CLL samples (N=5) ex vivo.

Analyses using patient PBMC samples (N=3) have shown that the CNL and SMAP combination triggers apoptotic cell death with cleavage of caspase9, capase3, and PARP in CLL cells, while normal T lymphocytes in these samples were relatively less affected. At the molecular level, mutually reinforcing interactions between SMAP and CNL were observed. Using mass-spectrometric profiling of sphingolipids, we discovered that PP2A activation by SMAP modulates sphingolipid metabolism, leading to enhanced accumulation of C6-ceramide as well as synthesis of endogenous, pro-apoptotic sphingolipid molecules (C14- and C16-ceramides) in cells exposed to CNL. In addition, co-treatment with SMAP and CNL induced synergistic/additive downregulation of pBAD and cMyc, suggesting cMyc and/or Bad-dependent pathways could serve as node of convergence for synergistic interaction seen in these cells.

In summary, we demonstrate that the CNL-SMAP combination displays exceptional synergy in CLL cells. At the molecular level, CNL and SMAP exhibit mutually reinforcing interactions that potentially converge on cMyc and Bad pathways, leading to synergistically increased apoptotic death in these cells. We intend to further characterize the role of cMyc and/or Bad pathway in synergistic interaction and assess the in vivo safety/efficacy of CNL-SMAP combination using a CLL xenograft mouse model. Collectively, this work provides the basis for further testing of this combination in in vivo models.

Williams:Pharmacyclics: Research Funding; Kymera: Consultancy, Research Funding; Astra Zeneca, Gilead, Kite Pharma, TG Therapeutics: Consultancy; International Oncology Network, Research to Practice: Honoraria; Janssen: Consultancy, Research Funding. Narla:RAPPTA Therapeutics: Consultancy, Other: Equity Ownership (Private company), Research Funding. Kester:Keystone Nano: Other: co-founder .