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A.A. Cortés et al.
respectively. In combination with nilotinib and pred- nisone, STAT5 and ERK were inhibited by 69.2±11.8% and 29.4±4.5%, respectively (Figure 3A and B). By con- trast, phosphorylation of AKT was not inhibited in any case (data not shown).
Ruxolitinib/nilotinib/prednisone combination decreases the synthesis of collagen I in bone marrow mesenchymal cells
Myelofibrosis is characterized by an increase in collagen deposition, among other fibrillar proteins, in BM, which prevents its proper functioning. To study the effect of rux- olitinib, nilotinib and prednisone (in monotherapy and in combination), on collagen expression, we utilized the HS27a mesenchymal cell line, together with TGF-β as an inductor of collagen expression. TGF-β increased the expression of COL1A1 200.9% over untreated cells (Figure 4A). Nilotinib decreased the mRNA expression of COL1A1 in HS27a cells by 60.2±0.9% in monotherapy, by 51.9±2.9% in combination with ruxolitinib and 62.2±1.9% in combination with ruxolitinib and pred- nisone. As a complementary test, we measured collagen expression by immunocytochemistry, finding that colla-
gen synthesis was reduced, especially in monotherapy (79.13±20.5%) and in combination with ruxolitinib (79.20±2.3%) (Figure 4B and Online Supplementary Figure S2).
Discussion
The management of MF remains challenging, even in the era of TKIs and personalized medicine. The discovery of the V617F mutation in JAK2 as a physiopathogenic mechanism of MPN3-7 prompted the development of JAK2 inhibitors and represented a revolution in the treat- ment of MF. Currently, the only approved JAK2 inhibitor for the treatment of MF and PV in the second-line is rux- olitinib,25 which has been shown to be effective in reduc- ing hepatosplenomegaly, resolving disease-related symp- toms, and producing a significant increase in overall sur- vival when compared with conventional therapies.26 Nevertheless, there are some limitations to the use of rux- olitinib, including hematologic toxicity (anemia and thrombocytopenia) and a failure to achieve histopatholog- ical and molecular complete responses.15 Accordingly, the
Table 3. Combination Index of ruxolitinib (R), nilotinib (N) and prednisone (P) in samples of cell lines. SET2: BA/F3 JAK2 wt or V617F cell lines were incubated with R, N, P or their combination for 48 hours and then Wst8 was performed.
Nilotinib (mM) 320
6.4
32
32
6.4
6.4
6.4
6.4
32
32
32
32 1.6 0.8
BAF3 JAK2 V617F SEM
BAF3 JAK2 wt
CI SEM P
0.192 0.090
0.086 ** 0.060 **
Ruxolitinib (nM)
6.4
Prednisone (mM) 0.16
CI Mean
>2
>2 >2 >2 0.067 0.198 0.082 0.095 0.049 0.040 0.051 0.063
CI Mean
0.815
0.596
1.374
0.857
0.538 0.448 0.396 0.337 0.155 0.158 0.173 0.153
P CI Mean 9.47 NA
1.6 0.32 1.6 0.32 0.32 1.6 1.6 0.32 0.32 1.6
3.35 NA 12.49 NA 187.51 NA
0.8 0.16 0.8 0.16 0.8 0.16
* NA ** NA *** 0.311 *** 0.172 *** NA *** 0.198 *** NA
*** 0.248 SEM P
0.261
0.189
0.144
0.109
0.162
0.057
0.089 *
0.040 *
0.015 ***
0.010 ***
0.011 ***
0.011 ***
0.02
0.12
0.03
0.03
0.02
0.01
0.01
Ruxolitinib (nM)
32
32 160 160 32 32 32 32 160 160 160
160
Prednisone (mM) 0.16
0.8 0.16 0.8 0.16 0.8 8 0.16
Nilotinib (mM) 1.6
SET2
0.01
8 1.6 8 1.6 1.6 8 8 1.6 1.6
8 0.8
NA: drugs combination with an effect less than of 20% compared to control. Combination Index (CI) < 0.9 indicates synergy (Italic); CI > 1.1 indicates antagonism (Italic Bold); CI from 0.9 to 1.1 indicates additivity (Bold). *P<0.05; **P<0.01; ***P<0.001.
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