Hsp90 inhibition in B cell acute lymphoblastic leukemia overcomes resistance to JAK2 inhibitors.

Purpose: The myeloproliferative neoplasm myelofibrosis is characterized by frequent deregulation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, and JAK inhibitors were shown to reduce splenomegaly and ameliorate disease-related symptoms. However, the mutant clone and bone marrow fibrosis persist in the majority of patients. Using preclinical models, we explored whether JAK and pan-deacetylase inhibitor combination yielded additional benefits.Experimental Design: The combination of the JAK1/2 inhibitor ruxolitinib and panobinostat was investigated using two different mouse models of JAK2 V617F -driven disease. A Ba/F3 JAK2 V617F cell-driven leukemic disease model was used to identify tolerated and efficacious doses. The drugs were then evaluated alone and in combination in a mouse model of myeloproliferative neoplasm-like disease based on transplantation of bone marrow transduced with a retrovirus expressing JAK2 V617F . Exposures were determined in blood and tissues, and phosphorylated STAT5 and acetylated histone H3 pharmacodynamic readouts were assessed in spleen and bone marrow. Histologic analysis was conducted on spleen and bone marrow, including staining of reticulin fibers in the latter organ.Results: The combination of ruxolitinib and panobinostat was found to have a more profound effect on splenomegaly, as well as on bone marrow and spleen histology, compared with either agent alone, and the analysis of pharmacodynamic readouts showed that ruxolitinib and panobinostat have nonoverlapping and complementary effects.Conclusion: Combining JAK1/2 and pan-deacetylase inhibitors was fairly well tolerated and resulted in improved efficacy in mouse models of JAK2 V617F -driven disease compared with the single agents. Thus, the combination of ruxolitinib and panobinostat may represent a promising novel therapeutic modality for myeloproliferative neoplasms.

798FN2 Background: Ruxolitinib (INC424), a potent and selective oral JAK1 and JAK2 inhibitor, has demonstrated rapid and durable reductions in splenomegaly and improved disease-related symptoms, role functioning, and quality of life in 2 phase 3 studies in patients with myelofibrosis. Panobinostat, a potent oral pan-deacetylase inhibitor (pan-DACi) that increases acetylation of proteins involved in multiple oncogenic pathways, has shown encouraging clinical activity in phase 1/2 trials of myelofibrosis. Here, we investigate the combination of ruxolitinib and panobinostat in mouse models of JAK2V617F-driven disease. Methods: Scid-beige female mice were randomized to treatment groups on the basis of baseline bioluminescence 4 days after injection of Ba/F3 EpoR JAK2V617F-luciferase cells. Mice were treated with vehicle control, panobinostat (4, 8, or 12 mg/kg intraperitoneal 3 times a week) alone or in combination with ruxolitinib (60 mg/kg orally twice daily), or ruxolitinib alone (n=7 each). Whole-body bioluminescence imaging was conducted on days 7 and 11. Spleens were weighed and extracts were obtained to determine levels of phosphorylated STAT5 (p-STAT5) and lysine acetylation. Results: Imaging on day 11 showed luminescence reductions (P <.05 compared with vehicle control) in all treatment groups (Table) A dose-dependent reduction was observed in mice treated with panobinostat alone. Combination therapy produced the greatest reductions in luminescence, as low as 3% of the untreated control in the ruxolitinib + panobinostat (12 mg/kg) group (P <.05 compared with the same dose of panobinostat alone or ruxolitinib alone). Similarly, reductions in spleen weight (P <.05 compared with control) were observed with ruxolitinib alone and panobinostat (12 mg/kg) alone and in all combination groups. Treatment with ruxolitinib reduced phosphorylation of the JAK2 downstream target STAT5, whereas panobinostat treatment reduced total STAT5 levels. Mice treated with both panobinostat and ruxolitinib had reduced levels of p-STAT5 and total STAT5 in spleen extracts. Lysine acetylation, unchanged in ruxolitinib-treated mice, was markedly increased in panobinostat-treated mice. Together, the pharmacodynamic analyses indicate that ruxolitinib and panobinostat are active and have nonoverlapping and complementary effects on biological pathways. There was no major change in tolerability, as assessed by body weight, between panobinostat alone and in combination with ruxolitinib. The experiment was repeated with the 2 top doses of panobinostat and ruxolitinib and showed similar results. Based on these encouraging data, the combination is currently being tested in a mouse JAK2V617F bone marrow transplant model of myeloproliferative neoplasm (MPN) disease to assess its disease-modifying potential. Conclusions: The combination of ruxolitinib and panobinostat showed significant improvements in anticancer activity compared with either treatment given alone in this JAK2V617F mouse model. Further studies in a murine MPN disease model are underway. Disclosures: Baffert: Novartis Pharma AG: Employment. Evrot:Novartis Pharma AG: Employment. Ebel:Novartis Pharma AG: Employment. Roelli:Novartis Pharma AG: Employment. Andraos:Novartis Pharma AG: Employment. Qian:Novartis Pharma AG: Employment. Romanet:Novartis Pharma AG: Employment. Murakami:Novartis Pharma AG: Employment. Radimerski:Novartis Pharma AG: Employment.

No abstract

<p>- PDF file 1093K, Supplementary Table S1. Activity and tolerability of ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, in a mouse model of Ba/F3 JAK2V617F cells-driven leukemic disease. Supplementary Table S2. Levels of panobinostat (PAN) and ruxolitinib (RUX) in blood and tissues post-final dose in a mouse model of Ba/F3 JAK2V617F cells-driven leukemic disease. Supplementary Table S3. Exposure to panobinostat (PAN) and ruxolitinib (RUX) in blood and tissues in a mouse model of Ba/F3 JAK2V617F cells-driven leukemic disease. Supplementary Table S4. Levels of panobinostat (PAN) and ruxolitinib (RUX) in blood and tissues post-final dose in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Table S5. Exposure to panobinostat (PAN) and ruxolitinib (RUX) in blood and tissues in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S1. Tolerability of ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, in a mouse model of Ba/F3 JAK2V617F cellsdriven leukemic disease. Supplementary Figure S2. Modulation of aberrant JAK2/STAT5 signaling in vitro and in vivo following treatment with ruxolitinib (RUX) and panobinostat (PAN), alone and in combination. Supplementary Figure S3. Efficacy and tolerability of ruxolitinib (RUX) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S4. Modulation of STAT5 phosphorylation in spleen and bone marrow following treatment with ruxolitinib (RUX) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S5. Histological analysis following treatment with ruxolitinib (RUX) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S6. Efficacy and tolerability of panobinostat (PAN) in a mouse model of JAK2V617F-driven MPN-like disease Supplementary Figure S7. Modulation of protein acetylation and acetylated histone H3 in spleen and bone marrow, respectively, following treatment with 21 panobinostat (PAN) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S8. Histological analysis following treatment with panobinostat (PAN) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S9. Efficacy and tolerability of ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, in a mouse model of JAK2V617Fdriven MPN-like disease. Supplementary Figure S10. Impact of ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, on relative levels of retrovirally transduced JAK2V617F in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S11. Histological analysis following treatment with ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, in a mouse model of JAK2V617F-driven MPN-like disease</p>

<div>Abstract<p><b>Purpose:</b> The myeloproliferative neoplasm myelofibrosis is characterized by frequent deregulation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, and JAK inhibitors were shown to reduce splenomegaly and ameliorate disease-related symptoms. However, the mutant clone and bone marrow fibrosis persist in the majority of patients. Using preclinical models, we explored whether JAK and pan-deacetylase inhibitor combination yielded additional benefits.</p><p><b>Experimental Design:</b> The combination of the JAK1/2 inhibitor ruxolitinib and panobinostat was investigated using two different mouse models of JAK2^V617F-driven disease. A Ba/F3 JAK2^V617F cell–driven leukemic disease model was used to identify tolerated and efficacious doses. The drugs were then evaluated alone and in combination in a mouse model of myeloproliferative neoplasm–like disease based on transplantation of bone marrow transduced with a retrovirus expressing JAK2^V617F. Exposures were determined in blood and tissues, and phosphorylated STAT5 and acetylated histone H3 pharmacodynamic readouts were assessed in spleen and bone marrow. Histologic analysis was conducted on spleen and bone marrow, including staining of reticulin fibers in the latter organ.</p><p><b>Results:</b> The combination of ruxolitinib and panobinostat was found to have a more profound effect on splenomegaly, as well as on bone marrow and spleen histology, compared with either agent alone, and the analysis of pharmacodynamic readouts showed that ruxolitinib and panobinostat have nonoverlapping and complementary effects.</p><p><b>Conclusion:</b> Combining JAK1/2 and pan-deacetylase inhibitors was fairly well tolerated and resulted in improved efficacy in mouse models of JAK2^V617F-driven disease compared with the single agents. Thus, the combination of ruxolitinib and panobinostat may represent a promising novel therapeutic modality for myeloproliferative neoplasms. <i>Clin Cancer Res; 19(22); 6230–41. ©2013 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> The myeloproliferative neoplasm myelofibrosis is characterized by frequent deregulation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, and JAK inhibitors were shown to reduce splenomegaly and ameliorate disease-related symptoms. However, the mutant clone and bone marrow fibrosis persist in the majority of patients. Using preclinical models, we explored whether JAK and pan-deacetylase inhibitor combination yielded additional benefits.</p><p><b>Experimental Design:</b> The combination of the JAK1/2 inhibitor ruxolitinib and panobinostat was investigated using two different mouse models of JAK2^V617F-driven disease. A Ba/F3 JAK2^V617F cell–driven leukemic disease model was used to identify tolerated and efficacious doses. The drugs were then evaluated alone and in combination in a mouse model of myeloproliferative neoplasm–like disease based on transplantation of bone marrow transduced with a retrovirus expressing JAK2^V617F. Exposures were determined in blood and tissues, and phosphorylated STAT5 and acetylated histone H3 pharmacodynamic readouts were assessed in spleen and bone marrow. Histologic analysis was conducted on spleen and bone marrow, including staining of reticulin fibers in the latter organ.</p><p><b>Results:</b> The combination of ruxolitinib and panobinostat was found to have a more profound effect on splenomegaly, as well as on bone marrow and spleen histology, compared with either agent alone, and the analysis of pharmacodynamic readouts showed that ruxolitinib and panobinostat have nonoverlapping and complementary effects.</p><p><b>Conclusion:</b> Combining JAK1/2 and pan-deacetylase inhibitors was fairly well tolerated and resulted in improved efficacy in mouse models of JAK2^V617F-driven disease compared with the single agents. Thus, the combination of ruxolitinib and panobinostat may represent a promising novel therapeutic modality for myeloproliferative neoplasms. <i>Clin Cancer Res; 19(22); 6230–41. ©2013 AACR</i>.</p></div>

<p>- PDF file 1093K, Supplementary Table S1. Activity and tolerability of ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, in a mouse model of Ba/F3 JAK2V617F cells-driven leukemic disease. Supplementary Table S2. Levels of panobinostat (PAN) and ruxolitinib (RUX) in blood and tissues post-final dose in a mouse model of Ba/F3 JAK2V617F cells-driven leukemic disease. Supplementary Table S3. Exposure to panobinostat (PAN) and ruxolitinib (RUX) in blood and tissues in a mouse model of Ba/F3 JAK2V617F cells-driven leukemic disease. Supplementary Table S4. Levels of panobinostat (PAN) and ruxolitinib (RUX) in blood and tissues post-final dose in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Table S5. Exposure to panobinostat (PAN) and ruxolitinib (RUX) in blood and tissues in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S1. Tolerability of ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, in a mouse model of Ba/F3 JAK2V617F cellsdriven leukemic disease. Supplementary Figure S2. Modulation of aberrant JAK2/STAT5 signaling in vitro and in vivo following treatment with ruxolitinib (RUX) and panobinostat (PAN), alone and in combination. Supplementary Figure S3. Efficacy and tolerability of ruxolitinib (RUX) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S4. Modulation of STAT5 phosphorylation in spleen and bone marrow following treatment with ruxolitinib (RUX) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S5. Histological analysis following treatment with ruxolitinib (RUX) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S6. Efficacy and tolerability of panobinostat (PAN) in a mouse model of JAK2V617F-driven MPN-like disease Supplementary Figure S7. Modulation of protein acetylation and acetylated histone H3 in spleen and bone marrow, respectively, following treatment with 21 panobinostat (PAN) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S8. Histological analysis following treatment with panobinostat (PAN) in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S9. Efficacy and tolerability of ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, in a mouse model of JAK2V617Fdriven MPN-like disease. Supplementary Figure S10. Impact of ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, on relative levels of retrovirally transduced JAK2V617F in a mouse model of JAK2V617F-driven MPN-like disease. Supplementary Figure S11. Histological analysis following treatment with ruxolitinib (RUX) and panobinostat (PAN), alone and in combination, in a mouse model of JAK2V617F-driven MPN-like disease</p>