Activated IL-6 signaling: A novel therapeutic target for CALR-mutated MPN

Mutations of the calreticulin gene (CALR^mut) are driver mutations known in the development of myeloproliferative neoplasms (MPN). CALR^mut helps to downstream Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signaling through interaction with, and activation of, the thrombopoietin receptor (MPL). Studies have also suggested that CALR expression in normal CD34⁺ cells increases as they differentiate to megakaryocytes (Mks). However, CALR has a wide-ranging action in protein and calcium homeostasis, and therefore, additional mechanisms may contribute to CALR^mut MPN. The MPN Hub recently published an article exploring the mechanism of CALR 52-bp deletion (CALR^del52), which can be found here.

In a newly published study in Blood Advances, Balliu et al,¹ investigated the mechanism contributing to CALR-mutated MPN through abnormal activation of the interleukin 6 (IL-6) pathway. The key findings are summarized below. 

Methods

In vitro and ex vivo experiments using cell lines included granulocyte/macrophage colony-stimulating factor (GM-CSF)-dependent UT7 and GM-CSF and thrombopoietin (TPO)-dependent UT7/mpl. CD34⁺ cells from healthy donors were collected. CD34⁺, UT7, and UT7/mpl cell lines and CALR type 1 (DEL) variants were used to generate CALR knockout (KO) cells. CRISPR/CRISPR associated protein 9 (Cas9) genome editing was used for CD34⁺ and green fluorescent protein-positive (GFP⁺) transfected cells. Chromatin immunoprecipitation (ChIP) assay was used to assess IL-6 promoter region chromatin occupancy.

Results

Development and characterisation of CALR DEL and KO cell lines

• UT7 and UT7/mpl CALR type 1-like (DEL) cells expressed (generated using CRISPR/Cas9), were as expected, and low levels of CALR DEL were observed despite CALR messenger RNA (mRNA) levels being comparable to CALR wild-type (CALR WT) parental cells suggesting instability of the mutated protein.
• Cytokine depleted CALR DEL and KO UT7 and UT/mpl cells showed extended survival and resistance to apoptosis compared with parental cells.

Abnormal activation of IL-6-signaling pathway in CALR DEL and KO mutant cells

• CD41⁺ and CD61⁺ cells increased by 1.9-fold and 2.1-fold, respectively, in CALR DEL and KO cells when induced with TPO (p < 0.001). Similar increases were seen in CD41⁺ and CD61⁺ (2.0-fold and 2.4-fold, respectively) in TPO-deprived conditions.
• Increased p-JAK1 and p-STAT3 levels in both CALR DEL and KO cells (5.6-fold and 7.95-fold and 4.3-fold and 5.3-fold, respectively) were also seen in TPO-deprived conditions, while p-JAK2 and p-STAT5 remained similar to WT cells.
• In comparison to WT cells, CALR DEL cells induced with TPO showed an increase in all pathways (p-JAK1, 7.2-fold; p-JAK2, 9.1-fold; p-STAT3, 2.3-fold; and p-STAT5, 1.8-fold). Similar changes in respective pathways were also seen in CALR KO cells (7.5-fold, 10-fold, 2.2-fold, and 1.9-fold, respectively). Overall, these findings indicate that CALR DEL mutation in the absence of WT protein induced autonomous JAK1/STAT3 activation independent of TPO, while JAK2/STAT5 signaling was preferentially induced by activation of MPL in either CALR DEL or KO cells.
• Increased amounts of IL-6 mRNA were maintained in both the absence (2.0-fold and 3.2-fold) and the presence (5.5-fold and 3.2-fold) of TPO in CALR DEL and KO UT7/mpl cells, respectively.
• IL-6R and glycoprotein 130 (gp130) on the cell membrane in CALR DEL and KO UT7/mpl cells were expressed at higher levels compared with WT cells.
• ChIP assay revealed enhanced interaction of STAT3 at the region −238 to −113, and specifically −143 to +48 in CALR DEL and KO cells. However, no significant expression and activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-_KB) in CALR DEL and KO cells were found.
• Exposure of IL-6 pathways to SC144 (chemical inhibitor of gp130), tocilizumab (TCZ; an IL-6R-blocking monoclonal antibody), and a monoclonal anti-IL-6 antibody, significantly reduced proliferation of CALR DEL and KO UT7/mpl cells by 58.3% ± 1.2% and 57.2% ± 3.6%, 33.4% ± 3.1% and 62.6% ± 7.2%, and 74.1% ± 3.4% and 54.4% ± 5.5%, respectively.

Activation of IL-6 signaling pathway is independent of MPL expression

• Compared with parental cells, CALR DEL and KO cells showed increased levels of IL-6 mRNA irrespective of being exposed (3.0-fold and 1.8-fold respectively) or not exposed (3.4-fold and 2.2-fold respectively) to GM-CSF, suggesting cell survival and proliferation.
• CALR DEL and KO UT7 cells were diminished by 63.9% ± 3% and 50% ± 4.1% when exposed to SC144, 69.1% ± 5.2% and 28.6% ± 4.1% with TCZ, and 58.8% ± 3% and 42.9% ± 4.1% with anti-IL-6 antibody, respectively.

CD34⁺ cells increase IL-6-dependent CFU-Mk generation

• CALR mRNA and protein content was reduced to half compared with mock cells, in sorted CD34⁺ cells. There was a significant increase in IL-6 mRNA levels (3.2-fold + 0.3-fold) in CALR KO CD34⁺ vs mock cells (p < 0.001).
• Megakaryocytic colony-forming units (CFU-Mks) were meaningfully increased by 10-fold in CALR KO CD34⁺ cells containing IL-6 compared with mock cells (p < 0.001), and decreased in IL-6-deprived conditions (38.2% + 4.3% vs 58% + 13.1%, respectively, p < 0.05). TCZ significantly reduced the number of CFU-Mk colonies from CALR KO CD34⁺ cells by 57.1% ± 2.7% (p < 0.001). 

CFU-MK colony generation inhibited by JAK1/2 and IL-6R inhibitor

• CFU-Mks generated from CD34⁺ cells of CALR- and JAK2^V617F-mutated patients were compared. In IL-6 deprived culture medium, a significant reduction was seen of CFU-Mks (−34.6% ± 2.8%, p <0.01), unlike CALR mutant or JAK2^V617F patients (−5.2% ± 1.0% and −1.90% ± 2.4%).
• Not only were the levels of IL-6 mRNA in CD34⁺ higher in CALR- (9.9-fold) and JAK2^V617F-mutated patients (10.6-fold, p < 0.01) compared with the control cells, but a majority (70% ± 10%) of CD34⁺ cells showed bright p-STAT3 immunofluorescence.
• The mean fluorescent intensity as well as the expression of gp130 and IL-6R on CD34⁺ from CALR- and JAK2V617F-mutated patients were much higher compared with the control cells. CD34⁺ gp130⁺ cells were 32.7% ± 1.6%, 42.4% ± 1.3%, and 22.9% ± 1.2%, and CD34⁺ IL-6R⁺ cells were 28.3% ± 2.7%, 29.5% ± 1.3%, and 13% ± 0.6%, in CALR-, JAK2^V617F-mutated patients, and controls, respectively.
• CD34⁺ cells from CALR- and JAK2^V617F-mutated patients were greatly inhibited with very low nanomolar concentrations of SC144, TCZ, and anti-IL-6 antibody.
• Colony-forming unit granulocyte/macrophage progenitor (CFU-GM) generated by CALR- and JAK2V617F-mutated CD34⁺ cells were largely inhibited by TCZ at 250 ng/mL by 51.67% ± 5.8% and 48.34% ± 5.1%, respectively. TCZ showed a minimal effect on burst-forming unit erythroid (BFU-E) compared with SC144 which was more effective.
• Finally, the combination of ruxolitinib (5 nM) and TCZ (5 and 50 ng/mL) reduced the CFU-Mks by 70.8% ± 2% and 90.8% ± 0.7% in CALR-mutated CD34⁺ cells, and by 79.5% ± 1.2% and 64.1% ± 4.5% in JAK2^V617F-mutated cells with a combination index of 0.12 and 0.30, respectively.

Conclusion

These data demonstrate the acquired cytokine independence, involvement in Mk differentiation, increased IL-6 mRNA levels and extracellular release of IL-6 accompanied with higher membrane-associated gp130 and IL-6R in cells, and increase in p-JAK1 and p-STAT3 levels. The study demonstrated an abnormal interaction between mutated CALR and gp130 and IL-6R. Combination of low nanomolar concentrations of TCZ and ruxolitinib led to lesser levels of CFU-Mks. Overall, there may be a potential for target inhibition of IL-6 signaling to have a therapeutic role in CALR- and possibly JAK2^V617F-mutated MPN. However, further evaluation is warranted to investigate the therapeutic efficacy of combined JAK2/IL-6 inhibition and paracrine effects of abnormal IL-6 pathway activation in MPN.