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In vitro assessment of Bcl-xL as a therapeutic target in Philadelphia-negative MPN

Sep 23, 2020

Molecular drivers of Philadelphia chromosome-negative (Ph ) myeloproliferative neoplasms (MPN) are mutations in Janus kinase 2 ( JAK2), calreticulin ( CALR) and myeloproliferative leukemia ( MPL) genes. One of the few options to target these driver mutations is treatment with JAK1/2 kinase inhibitors (JAKi) that have been shown to induce symptom relief and improved outcomes in patients with primary myelofibrosis (PMF) and polycythemia vera (PV). However, treatment with JAKi is not curative, and there is a remaining need to identify new targets for the treatment of MPN.

Jessica Petiti and colleagues published an article in Journal of Cellular Molecular Medicineon the evidence supporting the use of the B-cell lymphoma-extra large protein (Bcl-xL) as a marker of clinical severity and a potential target for therapy in Ph MPN. 1

Patients and methods

A total of 129 patients with MPN (36% essential thrombocythemia [ET], 36% PV, and 28% PMF) and 21 healthy volunteers were included. Out of the patients, 47% were JAK2wild-type and 53% carried the JAK2V1617F mutation.

For the in vitrostudies, a human erythroleukemic (HEL) cell line that was homozygous for the JAK2V1617F mutation was used.

Fluorescence-activated cell sorting was used to measure apoptosis in HEL cells and leukocytes from patients with MPN.

HEL cells and leukocytes isolated from patients were treated with ruxolitinib and ABT-737, a Bcl-xL inhibitor. The doses were determined by proliferation assay results and with reference to current literature.

Results

Protein and mRNA expression of Bcl-xL

mRNA expression was evaluated in patients with ET, PV, and PMF. There was a trend in increasing expression of the Bcl-xLgene moving from healthy controls and ET to PV and PMF, with the difference in expression being significant for the last two groups compared with the control (p < 0.001 for both). No difference in Bcl-xLexpression was found between leukocytes from controls and patients with ET. A similar pattern was found at the protein level, both in CD34 +hematopoietic progenitor cells and erythroblasts. These expression patterns were irrespective of the presence of the JAK2V617F mutation.

Splitting the groups into high and low expression showed a difference in distribution according to severity of disease in patients with Ph MPN. None of the healthy controls showed high Bcl-xLexpression, however 25% of the ET group, 55% of the PV group, and all of the PMF group showed high expression.

Synergism of inhibitors and impact on proliferation and apoptosis

Inhibition of JAK2 and Bcl-xL was hypothesized to have a synergistic effect, therefore, a proliferation assay in HEL cells was performed using ruxolitinib and ABT-737, alone and in combination. While IC50 (the half-maximal inhibitory concentration) for ABT-737 and ruxolitinib alone was 2.7 and 6.4 μmol/L, respectively, the IC50 for the combination decreased to only 0.8 μmol/L, indicating a strong synergistic inhibitory effect as confirmed in an isobologram analysis with a combination index of 0.44.

Furthermore, all three treatment groups showed an increase in the apoptotic fraction compared with the control group in HEL cells. ABT-737, alone or in combination, produced the most significant increase (33.7%, p < 0.01 and 57.4%, p < 0.001, respectively). Apoptosis and Bcl-xLexpression were strongly inversely correlated (r = –0.99, p < 0.001) in HEL cells.

These results were confirmed in leukocytes from patients with PV and PMF, demonstrating a 10% increase in apoptotic fraction with ruxolitinib only (p < 0.05) compared to 18.6% (p < 0.001) with ABT-737 alone. The strongest induction of apoptosis was seen with the combination, more than doubling the rate to 54.4% (p < 0.0001).

Bcl-xL expression in leukocytes of patients with PV and PMF

The impact of drug exposure on Bcl-xLmRNA expression was investigated in HEL cells. Interestingly, Bcl-xLexpression was already significantly downregulated by ruxolitinib alone, suggesting that expression or mRNA stabilization of this gene is dependent on the JAK2-STAT pathway. However, the treatment combination of ABT-737 and ruxolitinib produced the strongest decrease in Bcl-xLexpression (p < 0.01 vsABT-737 and p < 0.05 vsruxolitinib). These results were also confirmed at the protein level and indicate the presence of crosstalk between the two pathways.

In addition, mRNA expression of JAK2and Bcl-xLwere also analyzed along with JAK2 phosphorylation and Bcl-xL protein levels following the three treatment combinations in samples from patients with PV and PMF. Data showed that ruxolitinib and ABT-737 alone inhibited JAK2 phosphorylation, with only moderate impact on Bcl-xL. However, the combination of both drugs strongly downregulated Bcl-xL, at both the mRNA expression and protein levels.

Conclusion

Bcl-xL was identified as a marker showing strong correlation between expression levels and MPN subtype, with the highest expression seen in patients with PMF. In addition, Bcl-xL may serve as a potential therapeutic target, which can be inhibited by both the JAKi, ruxolitinib, and the Bcl-xL inhibitor, ABT-737. The synergistic role of a combined treatment with JAKi and Bcl-xL inhibition was supported by experiments showing maximum induction of apoptosis in both HEL cells and patient leukocytes when both drug classes were combined. Further investigation in in vivomodels is warranted.

  1. Petiti J, Lo Iacono M, Rosso V, et al. Bcl-xL represents a therapeutic target in Philadelphia negative myeloproliferative neoplasms.  J Cell Mol Med. 2020. Online ahead of print. DOI: 1111/jcmm.15730