The recurrent V617F mutation in JAK2 confers growth factor-independent proliferation for hematopoietic cells and plays an important role in the pathogenesis of myeloproliferative neoplasms (MPN). Absence of complete clinical remission in most MPN patients treated with the JAK1/2 inhibitor ruxolitinib highlights the need of new therapeutic strategies. Recent evidence indicates that IGF1R/IRS pathway is a potential target in MPN. In mice, AIRAPL down-regulation induces MPN transformation by upregulation of IGF1R signaling. Linsitinib (OSI-906) is a potent highly selective IGF1R/IR inhibitor, with anti-neoplastic effects in solid tumors, but underexplored in hematological neoplasms. We have previously noticed that OSI-906 reduces cell viability and inhibited IRS1/2, IGF1R, AKT1/2/3 and P70S6K in Ba/F3 JAK2V617F cells (EHA 2018 - Abstract #3288).
To investigate the in vivo and ex vivo effects of OSI-906 treatment in MPN phenotype and progenitors cells using JAK2V617F knockin-MPN mice.
For MPN phenotype induction, 5 × 106 bone marrow cells from JAK2V617F knockin mice were transplanted into lethally irradiated Pep boy mice. After 4 weeks, chimerism was evaluated by CD45.1 and CD45.2 markers (Becton-Dickinson) by flow cytrometry in peripheral blood. Mice with >70% CD45.2 cells were randomized and treated with vehicle (25 mM tartaric acid solution, n = 5) or OSI-906 (50 mg/kg, n = 4), both were administered oral by gavage, five days per week, from days 1 to 36. In day 37, animals were harvested and subjected to analysis of spleen and hematological parameters. Erythroid progenitors in the spleen and bone marrow were evaluated by CD71 and Ter119 markers. The immature subpopulation LSK (Sca1+/c-Kit+), the myeloid progenitor (Sca1−/c-Kit+), multipotent progenitors (MPP) (CD48+/C150−), long-term (LT-HSC) (CD48−/CD150+) and short-term (ST-HSC) (CD48−/150−) hematopoietic stem cells were evaluated by flow cytometry. All experiments were approved by the Animal Ethics Committee. Mann-Whitney test or ANOVA test and Bonferroni post-test was used. Alternatively, JAK2V617F knockin mice were euthanized between 10–16 weeks of age and the bone marrow was isolated, treated ex vivo with vehicle or OSI-906 (≥20 μM); and submitted to clonogenicity and to protein expression/activation studies.
OSI-906 treatment in vivo was well-tolerated by JAK2V617F-kockin MPN mice, but did not reduce the spleen volume. OSI-906 significantly reduced erythroid progenitors (p = 0.03) and increased myeloid progenitors (p = 0.01) in bone marrow when compared with vehicle. Hemoglobin, hematocrit and the number of MPP, ST-HSC, or LT-HSC were not modulated by OSI-906 treatment. Bone marrow cells from JAK2V617F knockin-MPN mice treated ex vivo with OSI-906 presented absence of colony formation, and decreased of protein expression/activation of IGF1R, AKT1/2/3, ERK1/2, and STAT3.
OSI-906 exhibited promising results ex vivo, including inhibition of IGF1R signaling pathways and progenitor cells proliferation. OSI-906 treatment in vivo in the current protocol reduced erythroid progenitors in bone marrow from JAK2V617F knockin-MPN mice. The lack of better results in vivo reinforces the need to adjust treatment regimen to this disease model.