In Vitro JAK1/2 Inhibition, in Association with Bortezomib and Lenalidomide, Is Comparable with Bortezomib, Lenalidomide and Dexametasone: An Alternative for Multiple Myeloma Patients with JAK2 overexpression?
Authors: Bleker de Oliveira Mariana , Fook Alves Veruska Lia , Eugenio Angela Isabel , Carlini Fernando Rodrigo , Yamamoto Mihoko , Colleoni Gisele W. B. 
Published: November 2019

Abstract
Background: Despite the absence of JAK1 and JAK2 mutations in multiple myeloma (MM), high levels of IL-6 constitutively activate the JAK/STAT pathway promoting survival and proliferation of tumor cells. Therefore, pharmacological inhibition of JAK proteins can be a potentially therapeutic strategy for myeloma treatment. Aims: 1) to identify expression of JAK1 and JAK2 in MM cell lines and in recently diagnosed MM patients; 2) to perform functional in vitro studies in MM cell lines treated with JAK/STAT pathway inhibitor (ruxolitinib), associated with drugs currently used in MM first line treatment (bortezomib, lenalidomide and dexamethasone), with and without co-culture with normal stromal cells; 3) to evaluate global gene expression of JAK/STAT pathway in cell lines treated with ruxolitinib to elucidate its mechanism of action in MM. Methods: JAK1 and JAK2 expression were analyzed in four cell lines (RPMI-8226, U266, SKO-007 and SKM-M2) and in bone marrow samples from 30 MM patients and 3 healthy controls by real time PCR. After IC50 calculation, drugs concentrations were: bortezomib (B) 10 nM for both RPMI-8226 and U266 cell lines; ruxolitinib (R) 30 µM for RPMI-8226 and 40 µM for U266 cell lines; lenalidomide (L) 10 µM for both cell lines; and dexamethasone (D) 1 µM for both cell lines. Apoptosis and cell cycle were evaluated by flow cytometry. PCR array for 92 JAK/STAT pathway related genes (Taqman® Array Human JAK/STAT Pathway, Applied Biosystems, Foster City, CA, USA) was performed in RPMI-8226 and U266 wild type and B+R treated cell lines, in duplicates. Results: Among the four cell lines, U266 presented the highest expression of JAK1 and JAK2 genes. JAK1 was overexpressed in 27% and JAK2 in 57% of 30 MM patients (considering at least 2-fold increase). After B+R treatment, RPMI-8226 showed increased number of cells in SubG0 phase (p<0.001) with reduction of cells in S (p<0.01) and G2/M (p<0.001) phases. In U266 cell line, there is a slight increase of cells in SubG0 phase (p<0.05). Also, after B+R treatment, both RPMI-8226 and U266 presented 50% of cells in late apoptosis, which was accompanied by reduction of expression levels of BCL-2 and BCL-XL anti-apoptotic genes. The expression profile of JAK/STAT pathway after B+R treatment showed that many JAK/STAT, Ras/Raf/MAPK and PI3K/Akt/mTOR pathways genes lost their expression, mainly in RPMI-8226, with insignificant changes in U266 expression pattern. Co-culture of RPMI-8226 with normal stromal cell line HS5 protected tumor cells from apoptosis, as the number of cells in late apoptosis decreased from 50% to 32% (p<0.001). The addition of immunomodulatory drug lenalidomide to the schedule (B+R+L) increased tumor cell death from 32% to 73% in co-culture (p<0.001). Despite the impressive results, B+R+L schedule was equivalent to currently used treatment for standard risk MM patients B+L+D (67% of cell death, p>0.05), in co-culture. Conclusion: B+R combination induced cell cycle arrest and apoptosis in U266 and RPMI-8226. The new drug combination B+R+L has in vitro results comparable with B+L+D and presents an alternative for MM treatment of almost 60% of cases bearing JAK2 overexpression. Our results support future studies using JAK inhibitors as an alternative for MM treatment in a Precision Medicine approach. Financial support: FAPESP 2010/17668-6 and CNPq.

Disclosures
No relevant conflicts of interest to declare.