Efficacy of the Pan-Bcl-2 Inhibitor (Obatoclax) As a Single Agent to Treat Myeloproliferative Neoplasm in JAK2V617F Murine Transplantation Model
Authors: Bianco Thiago Mantello , Coelho-Silva Juan L , Pereira-Martins Diego A , Scheucher Priscila Santos , Weinhäuser Isabel , Silva Antonio Bruno Alves , Silva Cleide Lucia , Mullally Ann , Kobayashi Susumu S , Rego Eduardo M , Traina Fabiola , Figueiredo-Pontes Lorena Lobo 
Published: November 2019

Background: The Jak2V617F mutation represents the most common molecular abnormality present in Philadelphia chromosome-negative Classical Myeloproliferative Neoplasms (Ph-neg MPNs). The differential expression of genes/proteins between the hematopoietic stem cells (HSC) and the more committed bone marrow progenitors may reveal, at least in part, the contribution of each cell type to the pathogenesis of the disease. Recently, it has been shown that the deregulation of molecules of the BCL2 family in MPN increases the survival of the transformed hematopoietic stem and progenitor cells (HSPCs), which may contribute the progression of the disease (Figueiredo-Pontes et al, Blood #122:2852 2013). In this context, it is possible that the inhibition of Bcl-2 proteins may increase the sensibility of transformed HSPC to apoptosis leading to the control of myeloid proliferation and potential reduction of the Jak2 mutation allele burden. To date, the treatment of Ph-neg MPNs remains non-curative despite the development of Jak2 inhibitors, which improve the clinical features but fail to eliminate leukemic initiating cells. Aim: Therefore, we aimed to study if the modulation of Bcl-2 proteins could have an impact in the control of MPN. Methods: Wild type CD45.1 C57BL/6 mice (age: 10-12 weeks) were transplanted with 5x106 total bone marrow from CD45.2 C57BL/6 mice (age: 10-12 weeks old) harboring the Jak2V617F mutation after lethal irradiation. All transplanted mice presented at least 80% of chimerism and developed MPN features (increased red blood cells (RBC), white blood cells (WBC) and platelets counts, elevated hematocrit and hemoglobin) 6 weeks post-transplantation. Treatment with vehicle (5% dimethylacetamide 0.5% methylcellulose, n=5) or Bcl-2 inhibitor (GX15-070 - the BH3 mimetic obatoclax 3 mg/Kg/day, n=6) once a day by oral gavage was started 7 weeks after transplant for a time period of 4 weeks. Spleen and bone marrow were collected from euthanized mice to evaluate both chimerism and frequency of HSC by using fluorescence-conjugated antibodies against CD45.1, CD45.2, Ter119, CD19, CD4, CD8 CD3, CD71, cKit, Sca-1, CD48, CD150, CD16/32 and CD34. Data are represented as means and standard error of mean (mean ± SEM). The statistical significance was defined as p≤0.05 and analyzed by the Mann Whitney test. Results: Mice treated with obatoclax exhibited a reduction of 32% in spleen weight compared to the vehicle group, but did not reach statistical significance (Control=0.19 ±0.01 g vs Obatoclax=0.13 ±0.03 g; p=0.3). No differences were observed in blood counts (RBC, WBC or platelets) between vehicle and obatoclax-treated mice. In addition, treatment with the pan-Bcl-2 inhibitor did not affect the number of Lineage negative, Sca-1+, c-Kit+ (LSK) cells (Control=5.8x104±2.8x104vs Obatoclax=4.7x104±1.2x104, p=0.9), MPP (Control=1.9x104±2.8x104vs Obatoclax=1.9x104±8.5x103, p=0.9), ST-HSC (Control=2.8x104±1.9x104vs Obatoclax=4.4x104±4x103, p=0.2) and LT-HSC (Control=9.8x103±4.7x103vs Obatocalx=1x104±2.4x103, p=0.9). Similarly, no differences were observed in the number of myeloid progenitors between the groups: (Control=6x105±1.7x105vs Obatoclax=1x106±1.9x105, p=0.1), MEP (Control=2.7x105±7.7x104vs Obatoclax=2.9x105±6.3x104,p=0.9), CMP (Control=9.8x104±2.1x104vs Obatoclax=2.3x105±4.4x104, p=0.06) and GMP (Control=1.5x105±6.5x104vs Obatoclax=3.6x105±8.8x104, p=0.08). The frequency of erythroid progenitors in the BM (Early progenitors, Control=18.0% ±3% vs Obatoclax=26.0%±1.4%, p=0.052, and late erythroid cells, Control=38.4%±9.5% vs Obatoclax=49.0%±2.7%, p=0.4) was also not altered by Bcl-2 inhibition. Conclusion: Our findings are in accordance with a phase II clinical trial demonstrating that obatoclax has no clinical activity in patients with Myelofibrosis. (Parikh et al, CLML 2010). Thus, we concluded that obatoclax did not reduce MPN burden in our model, when used as a single agent administrated orally. Yet, we cannot exclude the possibility that that the modulation of Bcl-2 overexpression at MPN initiating cells level could render these cells sensitive to the JAK2 inhibition. Hence, further studies will be necessary to assess whether the combination of obatoclax with other drugs such as JAK2 inhibitors could result in improved therapeutic response.

Disclosures
Mullally: Janssen: Research Funding. Kobayashi:Taiho Pharmaceutical: Research Funding; MiNA therapeutics: Research Funding; Pfizer: Consultancy; Ono Pharmaceutica: Consultancy; Chugai Pharmaceutical: Honoraria; Boehringer Ingelheim: Honoraria; Roche Diagnostic: Honoraria. Figueiredo-Pontes:Novartis: Honoraria.