Pharmacologic Inhibition of PI3Kδ Prolongs Survival of Mutant Shp2E76K-Expressing Mice
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Keywords:
Idelalisib
Juvenile myelomonocytic leukemia
Phosphoinositide 3-kinase
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STAT5
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Background
Gain-of-function mutations driving PI3K (phosphoinositide 3-kinase delta) enzyme activity lead to accumulation of senescent T cells, lymphadenopathy and immune-deficiency (PASLI disease), also known as Activated PI3K Syndrome (APDS). Inhibition of PI3K is a new therapeutic approach for the treatment of APDS.
Methods
We assessed leniolisib (CDZ173), a potent and selective PI3K inhibitor, in vitro for its effect on PI3K pathway activation induced by four APDS-causative p110δ variants in cell lines and in T cell blasts from patients. We then conducted a clinical trial with six APDS patients who completed a 12-week, open-label, multi-site, within-subject dose-escalation study of leniolisib to assess safety, pharmacokinetics and effects on lymphoproliferation and immune dysregulation.
Results
Expression of APDS mutant p110δ in cell lines and patient-derived lymphocytes led to increased pathway activity, measured as phosphorylation of AKT or S6, which was suppressed by leniolisib in a concentration dependent way. Oral leniolisib led to a dose-dependent reduction in PI3K/AKT pathway activity assessed ex vivo and resolved the immune dysregulation with normalization of circulating transitional and naive B cells and reduction in PD-1+CD4+ and senescent CD57+CD8+ T cells. After 12 weeks of treatment, all patients showed amelioration of lymphoproliferation with lymph node sizes and spleen volumes reduced by 39% (mean, range 26-57%) and 40% (mean, range: 13-65%), respectively.
Conclusions
Targeting hyperactive p110 in APDS patients using leniolisib was well tolerated and showed consistent laboratory and clinical improvement, including reduction in cellular immune dysfunction and lymphoproliferation (Funded by Novartis and NIAID DIR; ClinicalTrials.gov number, NCT02435173).
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Venetoclax
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Abstract Genistein is a soybean-derived isoflavone with antioxidant and anti-inflammatory effects. It also has tyrosine kinase inhibitory properties that attenuates proliferation of both normal and cancerous cells. Patients with severe congenital neutropenia (SCN) require continuous G-CSF treatment throughout their lifetime. Such prolonged treatment increases the incidence of myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) in this population. We hypothesize that genistein could counteract the deleterious effects of excessive hematopoietic stem cell (HSC) proliferation, which can lead to DNA damage, increase in radical oxygen species (ROS) production and DNA instability. To test our hypothesis we choose the p53 heterozygous (+/−) mouse model, once those animals have a constitutive enhancement in HSC proliferation. They also display high levels of ROS and DNA damage in comparison to wild type (WT). We started our experiments treating WT and p53+/− mice 3 times a week with subcutaneous injections of genistein. After treatment, we analyzed the total number of lin-ckit+sca+ (KLS) cells. The number of p53+/− KLS decreased while the WT KLS numbers remained unaffected. KLS cells were then analyzed for the amount of DNA damage via nuclear pH2AX staining. Our results show a 2 fold decrease in DNA damage on the p53+/− cells from treated animals, compared to untreated ones, while the levels of DNA damage remained unaltered on WT. Basal levels of ROS on p53+/− KLS cells were higher than in the WT KLS cells, but after treated with genistein those levels decreased by 10 fold. Interestingly, long term treatment with genistein did not result in a decrease of white blood cells on neither p53+/− nor WT animals. To elucidate the mechanism of action, KLS cells were sorted and cell cycle analyses showed that genistein inhibited cell cycle specifically in the stem cell population. Genistein reduced the percentage of p53+/− KLS cells in cycle, but it had no effect on WT cells. We then tested genistein effects on HSC population in bone marrow competitive repopulation assay. We transplanted lethally irradiated mice with a 50:50 mix of bone marrow from WT and p53+/− mice. After the transplant the animals were treated 3 times a week with subcutaneous injections of genistein or adjuvant. Animals were euthanized after 5 weeks of treatment and blood and bone marrow were analyzed. After genistein treatment, the percentages of p53+/− KLS cells in bone marrow were 60% on treated mice and 80% on untreated mice. This data suggests that genistein preferentially inhibits the highly proliferative stem cell population, indicating its preventive or therapeutic potential against the deleterious effects of excessive HSC proliferation. Understanding this mechanism of action could potentially reveal novel treatments for patients suffering with severe congenital neutropenia, leukemia, or other hematological malignancies with route on excessive HCS proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3489. doi:1538-7445.AM2012-3489
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Trichostatin A
Decitabine
Plerixafor
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Abstract Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN). The JAK2V617F mutation has been found in ~50% patients with MF. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. However, Ruxolitinib treatment has failed to induce disease remission or cure fibrosis. Therefore, there is a need to develop novel targeted therapies for MF. We found that PIM1 expression is significantly increased in mouse and human MPN/MF hematopoietic progenitor cells. Moreover, PIM1 knockdown markedly inhibited proliferation in JAK2V617F-expressing cells but not in wild-type JAK2-expressing cells. In this study, we investigated the efficacy of the second-generation pan-PIM kinase inhibitor, TP-3654, in hematopoietic cells expressing JAK2V617F and in a murine model of MF. We found that TP-3654 (0.5-1.0 μM) treatment significantly reduced proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F or human JAK2V617F-positive HEL and UKE-1 cells. TP-3654 Treatment also resulted in marked apoptosis in Ba/F3-EpoR-JAK2V617F, HEL and UKE-1 cells but not in wild-type JAK2 expressing Ba/F3-EpoR cells. Additionally, TP-3654 and Ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, TP-3654 significantly inhibited MPN/MF CD34+ hematopoietic progenitor colony growth. We previously reported the generation of JAK2V617F knock-in mice. Whereas heterozygous JAK2V617F knock-in mice exhibit a polycythemia vera phenotype, mice expressing homozygous JAK2V617F rapidly develop high-grade MF. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of TP-3654 alone or in combination with Ruxolitinib against MF. Treatment of TP-3654 alone significantly reduced the increase in white blood cell (WBC) and neutrophil counts as well as spleen size in mice expressing homozygous JAK2V617F compared with vehicle treatment. Combined treatment of TP-3654 and Ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow and spleens of TP-3654-treated mice whereas Ruxolitinib treatment did not significantly reduce fibrosis. Combined TP-3654 and Ruxolitinib treatment almost completely eliminated fibrosis in the bone marrow and spleens of homozygous JAK2V617F mice. TP-3654 treatment was well tolerated and did not cause any significant toxicity in wild type mice. RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells from these drug-treated mice revealed that the genes related to TNFα and WNT signaling pathways were significantly down-regulated by treatment of TP-3654 alone or TP-3654 and Ruxolitinib combination compared with vehicle. Overall, our results suggest that TP-3654 alone or in combination with Ruxolitinib may have therapeutic potential for treatment of MF. Citation Format: Avik Dutta, Dipmoy Nath, Yue Yang, Golam Mohi. The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1874.
Ruxolitinib
Erythropoietin receptor
Myeloproliferative neoplasm
Interleukin 3
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