The Bone Marrow-Mediated Protection of Myeloproliferative Neoplastic Cells to Vorinostat and Ruxolitinib Relies on the Activation of JNK and PI3K Signalling Pathways

Abstract

The classical BCR-ABL-negative Myeloproliferative Neoplasms (MPN) are a group of heterogeneous haematological diseases characterized by constitutive JAK-STAT pathway activation. Targeted therapy with Ruxolitinib, a JAK1/2-specific inhibitor, achieves symptomatic improvement but does not eliminate the neoplastic clone. Similar effects are seen with histone deacetylase inhibitors (HDACi), albeit with poorer tolerance. Here, we show that bone marrow (BM) stromal cells (HS-5) protected MPN-derived cell lines (SET-2; HEL and UKE-1) and MPN patient-derived BM cells from the cytotoxic effects of Ruxolitinib and the HDACi Vorinostat. This protective effect was mediated, at least in part, by the secretion of soluble factors from the BM stroma. In addition, it correlated with the activation of signalling pathways important for cellular homeostasis, such as JAK-STAT, PI3K, JNK, MEK-ERK and NF-κB. Importantly, the pharmacological inhibition of JNK and PI3K pathways completely abrogated the BM protective effect on MPN cell lines and MPN patient samples. Our findings shed light on mechanisms of tumour survival and may indicate novel therapeutic approaches for the treatment of MPN.

Fig 1. HS-5 stromal cells protect SET-2 cells from Vorinostat and Ruxolitinib- induced apoptosis.

SET-2 cells were cultured in vitro (no stroma) and co-cultured with a stromal layer of HS-5 cells (+ HS-5), HS-5 conditioned media [+ CM (HS-5)] and or separated by a 0.4-μm-thick micropore membranes [+ HS-5 (+ TW)]. SET-2 cells were incubated in these conditions for 72h and treated with the indicated concentrations of Vorinostat (A and C) and Ruxolitinib (B and D). At 72h of co-culture, SET-2 cells were harvested, stained with CD45 (to distinguish between SET-2 and the stromal cell lines) and Annexin-V/PI (A and B) and PI alone (C and D) to determine cellular viability by Flow Cytometry analysis. The graphs indicate Viability Index that normalizes the viability values to those of the control conditions (A and C—0.0μM Vorinostat and B and D—0nM Ruxolitinib). Values indicate the mean ± standard deviation of the eight (A and B) and three (C and D) experiments performed (* 0.05>p; ** 0.01>p; *** 0.001>p).

Fig 2. HS-5 stroma activates crucial signalling pathways in SET-2 cells.

SET-2 cells were cultured in vitro (no stroma), co-cultured in a stromal layer of HS-5 cells (+ HS-5) and with HS-5 conditioned media [+ CM (HS-5)] at the indicated time point the cells. Cells were lysed and the phosphorylation and total levels of STAT5, STAT3, JNK/SAPK, ERK1/2, Akt/PKB, GSK3α/β, S6 and p65/RELA were analyzed by immunoblot. Actin was used as loading control. The data is representative of two independent experiments.

Fig 3. Pharmacological inhibition of JNK and PI3K signalling pathways completely reverted HS-5 BM stroma mediated protection of SET-2 cells.

(A and B) SET-2 cells were cultured in vitro (no stroma) and co-cultured in a stromal layer of HS-5 cells (+ HS-5) for 72h in the presence of 2.0μM Vorinostat (Vor.), 500nM Ruxolitinib (Rux.), 5μM and 10μM SP600125 (JNK inhibitor) (in A) and 5μM and 10μM LY294002 (PI3K inhibitor) (in B). After 72h, the SET-2 cells were harvested, stained with CD45 (to distinguish between SET-2 and the stromal cell lines) and Annexin-V/PI or PI alone to determine cellular viability by Flow Cytometry analysis as described in “Materials and Methods”. The panels in (A and B) show the Viability Index graphs that normalize the viability values to those of the control condition (non-treated condition NT). Values indicate the mean ± standard deviation of the five experiments performed (* 0.05 >p; ** 0.01>p; *** 0.001>p).

Fig 4. Pharmacological inhibition of JNK and PI3K signalling pathways abrogate the HS-5 BM stroma induced protection of patient derived BM Lin^-CD34⁺ cells from Vorinostat–induced cytotoxicity.

Lin^-CD34⁺ cells were isolated as described in “Material and Methods” section and co-cultured in a stromal layer of HS-5 cells for 72h in presence of the indicated concentrations of Vorinostat (A and B), Ruxolitinib (C and D) SP600125 (JNKi–A and C) and LY294002 (PI3Ki–B and D). At 72h of co-culture, cells were harvested, stained with CD45 (to distinguish between Lin^-CD34⁺ and HS-5 cells) and Annexin-V/PI to determine cellular viability by Flow Cytometry analysis as described in “Materials and Methods”. The results are representative of nine MPN patients tested and graphics indicate the percentage of viable cells (Annexin-V and PI negative cells) in the indicated conditions (* 0.05 >p; *** 0.001>p).

Fig 5. Model for the proposed BM mediated protection of MPN cells.

In vitro, MPN cells (SET-2, HEL, UKE-1 cell lines and MPN patient BM-derived Lin^-CD34⁺ cells) are killed once exposed to chemotherapeutic agents, like Vorinostat and Ruxolitinib, as a result of the inhibition of HDAC enzymes and JAK2V617F mutant protein kinase, respectively. However, in vivo (in MPN patients) or in the co-culture assays with the HS-5 BM stroma, MPN cells are able survive the cytotoxic action of these noxious agents through the activation of the JNK and PI3K signalling pathways, allowing MPN cells to escape these chemotherapeutic regiments and prolonging disease in MPN patients. The mechanisms underlying the activation of these signalling pathways are still elusive, but we postulate that involves the secretion of soluble factors by the BM stroma and the direct interaction between the MPN cells and the BM microenvironment.