Mycosis fungoides (MF) is the most common form of CTCL. The peripheral blood of MF patients is variably involved with malignant skin-homing T cells, with Sézary syndrome representing an advanced form characterized by erythroderma and frank leukemic expansion of the cells.
The Class 1A phosphoinositide-3-kinase (PI3K) signaling cascade is involved in control of a variety of cellular responses including cell-cycle and anti-apoptotic pathways. Inappropriate activation / phosphorylation of the downstream effector, Akt, via dysregulated PI3K signaling has been detected in a variety of cancers including CTCL, and may contribute to proliferation and survival of malignant cells. While the α and β isoforms of PI3K are ubiquitous in their distribution, expression of δ and γ is majorly associated with immune cells including T-lymphocytes. Selective pharmacologic targeting of PI3K δ/γ may therefore have therapeutic potential. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor. RP6530 demonstrates high potency against PI3Kδ (IC50=25 nM) and γ (IC50=33 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency has been confirmed in target-specific assays, namely anti-FcεR1-(EC50=38 nM) or fMLP (EC50=39 nM) induced CD63 expression in human whole blood basophils, LPS-induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS-induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). In this study, we examined the effects of RP6530 on Akt phosphorylation in CTCL cell lines HH and Sez4 and in purified malignant T cells from six CTCL patients exhibiting Sézary Syndrome.
Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Malignant T cells were isolated by negative selection using an antibody plus magnetic bead kit (Miltenyi Biotec) designed for CD4+ T cell isolation supplemented with patient-specific anti-CD7 and/or CD26 antibodies. Purity of isolated cells as tested by flow cytometry was >96%. Purified malignant T cells were cultured in RPMI+1% BSA overnight then treated ± inhibitor for 1.5 hr, with serum plus a cytokine mix (IL2+7+15) added for the final 30 minutes to maximize Akt phosphorylation. The ratio of phosphorylated, pSer473-Akt to total Akt was then measured by phospho-flow cytometry.
RP6530 caused dose-dependent inhibition of Akt Ser473 phosphorylation, with significant inhibition detectable even at the lowest concentration tested (10 nM, P = 0.035) and reaching 39.29% at 1000 nM. Inhibition observed was significantly greater (P = 0.0133) than that seen with the potent PI3K control inhibitor LY294002 (27.50%) at the same concentration. Non-linear regression analysis (Prism 5.0) of the mean pSer473-Akt:total-Akt ratio indicated the IC50 for RP6530 (198.2 nM) at ∼4-fold lower than for LY (939.7 nM).
In addition, apoptosis induction as measured by Annexin V binding, was tested in purified malignant T cells from four Sezary Syndrome patients. Following a 48hr incubation with RP6530, a dose-dependent increase in late apoptotic, Annexin V+ Propidium Iodide (PI)+, cells was seen. The compound was effective even at lower concentrations, with 60.15% more Annexin V+ PI+ cells seen in cultures treated with 100 nM inhibitor than untreated cultures (P= 0.027), and with a 120.43% increase seen at the highest concentration tested (10 μM).
Results from in vitro studies suggest the therapeutic potential of RP6530 in the treatment of CTCL with peripheral blood involvement. Findings provide a rationale for future clinical trials in T-cell malignancies.
Girardi:Rhizen Pharmaceuticals S.A.: Research Funding. Vakkalanka:Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership. Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.
